Utilization of starch for biological production by fermentation

ABSTRACT

This invention relates to a method for utilizing less purified starch in fermentation processes. One example is a recombinant  E. coli  containing a exogenous extracellular isoamylase activity that is capable of utilizing small oligomers containing (1,6) linkages (including but not limited to isomaltose and panose) in fermentations to produce useful products. The invention is useful in large-scale industrial biofermentations by reducing the cost of the substrate carbohydrate.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/405,896, filed Aug. 23, 2002.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of molecular biology.More specifically it describes microbial hosts containing genes thatexpress enzymes that effectively convert starch products into afermentation product.

BACKGROUND OF THE INVENTION

[0003] Fermentation is an important technology for the biocatalyticconversion of renewable feedstocks into desirable products.Carbohydrates are traditional feedstocks in the fermentation industry.It is often the case that carbohydrates used as a substrate contributemore to the cost of manufacture than any other single component.Depending on the particular process, from 25 to 70% of the total cost offermentation may be due to the carbohydrate source. (Crueger andCrueger, Biotechnology: A Textbook of Industrial Microbiology, SinauerAssociates: Sunderland, Mass., pp 124-174 (1990); Atkinson and Mavituna,Biochemical Engineering and Biotechnology Handbook, 2^(nd) ed.; StocktonPress: New York, pp 243-364 (1991)). For such economic reasons, highlypurified glucose or sucrose can seldom be used as a substrate.

[0004] Starch, a carbohydrate, is a mixture of two differentpolysaccharides each consisting of chains of linked, repeatingmonosaccharide (glucose) units. The mixture consists of two separatepolysaccharides, amylose and amylopectin. Amylose is a linearpolysaccharide with glucose units connected exclusively through α(1,4)glycosidic linkages. Glucose units in amylopectin are also linkedthrough α(1,4) glycosidic linkages, and additionally are linked throughα(1,6) glycosidic linkages, about one every 30 glucose residues. Theratio of amylopectin to amylose in starch varies from one plant speciesto another, but is generally in the range of 3-4 to 1 (Kainuma, pp125-150 in Starch; Whistler, Bemiller, and Pashcall eds., AcademicPress, Orlando, Fla. (1984)).

[0005] Commercial starch is produced primarily through the wet millingprocess. The final products from a wet mill, however, include verylittle unprocessed starch. By far, the majority of products made are inthe form of fully processed starch (monosaccharides, including glucose)or smaller degradation products derived from starch. Typically, anamylase enzyme is used to break starch into smaller chains (Blanchard,Technology of Corn Wet Milling (1992), Elseiver, Amsterdam, TheNetherlands, pp. 174-215). Various commercial sources of α-amylaseexist, but, regardless of enzyme source, reaction products are generallythe same with respect to size and linkage-type. Amylase digestion ofstarch results in a product known as a limit dextrin that includes smallstarch chains containing 2-10 glucose units (oligosaccharides). Becauseamylase cannot hydrolyze the α(1,6) glycosidic linkages in amylopectin,limit dextrins contain both α(1,4)- and α(1,6)-linked glucoseoligosaccharides. Alternatively, raw starch may be treated bynon-enzymatic means (for example, by acid hydrolysis) to produce starchproducts substantially similar to limit dextrin.

[0006] In the wet milling industry, limit dextrins are further processedinto glucose for use as a carbon source for fermentations to producevarious chemicals, commercial enzymes, or antibiotics. Relatively pureglucose is preferred as a carbohydrate source when the popularbiocatalyst, Escherichia coli, is used in the fermentation process. Thisis because E. coli does not utilize components of limit dextrins (i.e.,panose, isomaltose, and high molecular weight oligosaccharides withchains larger than about ten α(1,4)-linked glucose units) that arecommonly contained in alternate low-cost fermentation media (Lin,Escherichia coli and Salmonella typhimuium, pp. 245-265, Neidhardt, ed.;American Society for Microbiology, Washington, D.C. (1987)). Glucoseoligomers containing α(1,6)-linkages are not transported into the celland E. coli does not produce an enzyme that degrades this material whensupplied extracellularly (Palmer et al., Eur. J. Biochem. 39:601-612(1973)).

[0007] Making relatively pure glucose from starch that is suitable foruse by E. coli requires many process steps and additional enzymes,adding significantly to the cost of product manufacture.

[0008] Thus, the problem to be solved is the lack of a process toutilize low-cost starch products in large-scale fermentative productionprocesses. An ability to more completely ferment low cost, partiallydegraded starch would lower the cost of manufacture for products madethrough fermentation.

SUMMARY OF THE INVENTION

[0009] Applicants have provided an isolated nucleic acid moleculeencoding an α(1,6)-linked glucose oligosaccharide hydrolyzing enzymeselected from the group consisting of: (a) an isolated nucleic acidmolecule encoding the amino acid sequence selected from the groupconsisting of SEQ ID NOs:2, 4, and 6; (b) a nucleic acid molecule thathybridizes with (a) under the following hybridization conditions:0.1×SSC, 0.1% SES, 65° C. and washed with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS; and (c) a nucleic acid molecule that is complementaryto (a) or (b).

[0010] Applicants have provided nucleic acid compositions comprisingcoding regions for a signal peptide and an α(1,6)-linked glucoseoligosaccharide hydrolyzing enzyme such that a chimeric protein isexpressed that directs the hydrolyzing activity external to thecytoplasm (extracellularly). The isolated nucleic acid molecule mayencode a signal peptide as set forth in SEQ ID NO:24 or SEQ ID NO:25.The nucleic acid sequence of the signal sequence is SEQ ID NO:26 or SEQID NO:27. The isolated nucleic acid molecule may encode an α(1,6)-linkedglucose oligosaccharide hydrolyzing polypeptide as set forth in SEQ IDNOs:2, 4, 6, 17, or 31.

[0011] Applicants have provided recombinant organisms comprising anα(1,6)-linked glucose oligosaccharide hydrolyzing enzyme that enablesthe utilization of exogenously added α(1,6)-linked glucoseoligosaccharides (e.g., isomaltose and panose) for the fermentativeproduction of useful products. The α(1,6)-linked glucose oligosaccharidehydrolyzing polypeptide may be selected from SEQ ID NO:2, SEQ ID NO:6,SEQ ID NO:17, or SEQ ID NO:31. The invention also encompasses anα(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide encoded bythe nucleic acid molecule set forth in SEQ ID NOs:1, 3, 5, 16, or 30.The invention also includes isolated nucleic acid molecules selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO:28, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42.The invention also includes the polypeptide SEQ ID NO:4, SEQ ID NO:29,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,and SEQ ID NO:43.

[0012] The invention also encompasses a chimeric gene comprising theisolated nucleic acid molecules set forth herein operably linked tosuitable regulatory sequences. The suitable regulatory sequence isselected from the group comprising CYC1, HIS3, GAL1, GAL10, ADH1, PGK,PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, AOX1, lac, ara, tet, trp,IP_(L), IP_(R), T7, tac, trc, apr, npr, nos, and GI. The inventionencompasses transformed host cells wherein the chimeric gene isintegrated into the chromosome or is plasmid-borne.

[0013] Applicants have also provided a method for degrading limitdextrin comprising:

[0014] (a) contacting a transformed host cell comprising:

[0015] (i) a nucleic acid molecule encoding the enzymes selected fromthe group consisting of SEQ ID NOs:2, 6, 17 and 31;

[0016] (ii) a nucleic acid molecule that hybridizes with (i) under thefollowing hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washedwith 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or

[0017] (iii) a nucleic acid molecule that is complementary to (i) or(ii),

[0018] with an effective amount of limit dextrin substrate undersuitable growth conditions; and

[0019] (b) optionally recovering the product of step (a).

[0020] The invention also encompasses a method for producing a targetmolecule in a recombinant host cell comprising: contacting a transformedhost cell comprising: (i) an isolated nucleic acid molecule encoding achimeric protein comprised of a signal peptide linked to anα(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide; (ii) anucleic acid molecule that hybridizes with (i) under the followinghybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or (iii) a nucleic acidmolecule that is complementary to (i) or (ii); and a chimeric gene forconverting mononsaccharides to the target molecule, in the presence oflimit dextrin under suitable conditions whereby the target molecule isproduced; and optionally recovering the target molecule produced. Thesignal peptide may be selected from SEQ ID NO:24 or SEQ ID NO:25. Theα(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide may beselected from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17 or SEQ ID NO:31.The transformed host cell may be selected from bacteria, yeast orfilamentous fungi. This invention includes producing 1,3 propanediol,glycerol, and cell mass from limit dextrin.

[0021] The invention also encompasses a polypeptide having an amino acidsequence that has at least 69% identity based on the BLASTP method ofalignment when compared to a polypeptide having the sequence as setforth in SEQ ID NO:17, the polypeptide having an α(1,6)-linked glucoseoligosaccharide hydrolyzing activity.

BRIEF DESCRIPTION OF THE DRAWINGS, BIOLOGICAL DEPOSITS, AND SEQUENCEDESCRIPTIONS

[0022]FIGS. 1a through 1 d show the results of the E. coli strain DH5acontaining the plasmids pUC18 (FIG. 1a) (negative control) and pUC18containing the mature coding sequence from the clones j20 (FIG. 1b), k1(FIG. 1c), or h12 (FIG. 1d). Total protein extracts were isolated fromsonicated cells and incubated with panose (250 μg/ml) at 37° C. for twohours. A high performance anion exchange chromatogram of the productsafter digestion is shown.

[0023] Applicants made the following biological deposits under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for the Purposes of Patent Procedure at the AmericanType Culture Collection (ATCC) 10801 University Boulevard, Manassas, Va.20110-2209: Depositor Identification Int'l. Depository ReferenceDesignation Date of Deposit Escherichia coli RJ8n ATCC PTA-4216 9 Apr.2002

[0024] The listed deposit(s) will be maintained in the indicatedinternational depository for at least thirty (30) years and will be madeavailable to the public upon the grant of a patent disclosing it. Theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentaction.

[0025] Applicants provide a sequence listing containing 43 sequences.The sequences are in conformity with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) andconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of theAdministrative Instructions) and with the corresponding United StatesPatent and Trademark Office Rules set forth in 37 C.F.R. §1.822. GeneSEQ ID SEQ ID ORF Name Name Base Peptide Strain of Originmbc1g.pk007.h12 algB 1 2 Bifidobacterium breve mbc2g.pk018.j20 algA 3 4Bifidobacterium breve mbc1g.pk026.k1 algA 5 6 Bifidobacterium breve dexBdexB 16 17 Streptococcus mutans

[0026] SEQ ID NOs:1-6 are nucleic and amino acid sequences of threegenes/gene products obtained from Bifidobacterium breve ATCC 15700.

[0027] SEQ ID NOs:7-15 and 18-23 are primers for PCR.

[0028] SEQ ID NOs:16-17 are nucleic and amino acid sequences disclosedin public databases for Streptococcus mutans (ATCC 25175D).

[0029] SEQ ID NO:24 is the amino acid sequence for the native signalpeptide from the Bifidobacterium breve gene, mbc2g.pk018.j20 (alsocontained within SEQ ID NO:3).

[0030] SEQ ID NO:25 is the amino acid sequence for the non-native signalpeptide used to target enzymes coded for by the Bifidobacterium brevembc1g.pk026.k1 and Streptococcus mutans dexB genes.

[0031] SEQ ID NO:26 is the nucleic acid sequence for the Bifidobacteriumbreve gene mbc2g.pk018.j20 signal peptide

[0032] SEQ ID NO:27 is the nucleic acid sequence for the Bacillussubtilis neutral protease gene signal peptide.

[0033] SEQ ID NO:28 is the nucleic acid sequence for the Bifidobacteriumbreve gene mbc2g.pk018.j20 signal peptide linked to the coding sequencefor the Bifidobacterium breve mbc2g.pk018.h12 gene.

[0034] SEQ ID NO:29 is the amino acid sequence for the Bifidobacteriumbreve gene mbc2g.pk018.j20 signal peptide linked to the amino acidsequence for the Bifidobacterium breve mbc2g.pk018.h12 gene.

[0035] SEQ ID NO:30 is the nucleic acid sequence for the Bifidobacteriumbreve gene mbc2g.pk018.j20 without its native signal peptide sequence.

[0036] SEQ ID NO:31 is the amino acid sequence for the Bifidobacteriumbreve gene mbc2g.pk018.j20 without its native signal peptide sequence.

[0037] SEQ ID NO:32 is the nucleic acid sequence for the Bifidobacteriumbreve gene mbc2g.pk018.j20 signal peptide linked to the coding sequencefor the Bifidobacterium breve mbc2g.pk018.k1 gene.

[0038] SEQ ID NO:33 is the amino acid sequence for the Bifidobacteriumbreve gene mbc2g.pk018.j20 signal peptide linked to the amino acidsequence for the Bifidobacterium breve mbc2g.pk018.k1 gene.

[0039] SEQ ID NO:34 is the nucleic acid sequence for the Bifidobacteriumbreve gene mbc2g.pk018.j20 signal peptide linked to the coding sequencefor the Streptococcus mutans dexB gene.

[0040] SEQ ID NO:35 is the amino acid sequence for the Bifidobacteriumbreve gene mbc2g.pk018.j20 signal peptide linked to the amino acidsequence for the Streptococcus mutans dexB gene.

[0041] SEQ ID NO:36 is the nucleic acid sequence for the Bacillussubtilis neutral protease gene signal peptide linked to the codingsequence for the Bifidobacterium breve mbc2g.pk018.h12 gene.

[0042] SEQ ID NO:37 is the amino acid sequence for the Bacillus subtilisneutral protease gene signal peptide linked to the amino acid sequencefor the Bifidobacterium breve mbc2g.pk018.h12 gene.

[0043] SEQ ID NO:38 is the nucleic acid sequence for the Bacillussubtilis neutral protease gene signal peptide linked to the codingsequence for the Bifidobacterium breve mbc2g.pk018.j20 gene.

[0044] SEQ ID NO:39 is the amino acid sequence for the Bacillus subtilisneutral protease gene signal peptide linked to the amino acid sequencefor the Bifidobacterium breve mbc2g.pk018.j20 gene.

[0045] SEQ ID NO:40 is the nucleic acid sequence for the Bacillussubtilis neutral protease gene signal peptide linked to the codingsequence for the Bifidobacterium breve mbc2g.pk018.k1 gene.

[0046] SEQ ID NO:41 is the amino acid sequence for the Bacillus subtilisneutral protease gene signal peptide linked to amino acid sequence forthe Bifidobacterium breve mbc2g.pk018.k1 gene.

[0047] SEQ ID NO:42 is the nucleic acid sequence for the Bacillussubtilis neutral protease gene signal peptide linked to the codingsequence for the Streptococcus mutans dexB gene.

[0048] SEQ ID NO:43 is the amino acid sequence for the Bacillus subtilisneutral protease gene signal peptide linked to amino acid sequence forthe Streptococcus mutans dexB gene.

DETAILED DESCRIPTION OF THE INVENTION

[0049] Applicants have solved the stated problem. The present inventionprovides several enzymes that, when expressed in a production host,enable the host to utilize α(1,6)-linked glucose oligosaccharides, whichare components of low cost starch products. The invention also providessignal sequences that enable α(1,6)-linked glucose oligosaccharidehydrolyzing enzymes to be targeted extracellularly.

[0050] Low cost starch products are obtained, for example, from theaction of commercially available amylase enzymes on raw starch and otherfeed stocks containing α(1,6)-linked glucose oligosaccharides to producea limit dextrin. The efficient use of the low cost starch productsrequires genetically engineering a host organism (for example, E. coli),such that the recombinant organism produces an enzyme that degradesα(1,6)-linked glucose oligosaccharides. Enzymes that degradeα(1,6)-linked glucose oligosaccharides are known (Vihinen and MantsalaCrit. Rev. in Biochem. Mol. Biol. 4:329-427 (1989)). Further, enzymesthat degrade these linkages are known to be present both intracellularly(within the cytoplasm) and extracellularly (external to the cytoplasm)in their native state.

[0051] Where a host organism lacks a transport system, engineering anintracellular enzyme to have access to limit dextrin (or otherfeedstocks containing α(1,6)-linked glucose oligosaccharides) suppliedexternally may be accomplished by adding a native or non-native signalpeptide. Signal peptides enable the α(1,6)-linked glucoseoligosaccharide degrading protein to be directed to an extracellularlocation (external to the cytoplasm), and give access to substrates nottaken into the cell (Nagarajan et al., Gene 114:121-126 (1992)).Examples of signal peptides that translocate protein across the cell'smembrane include, but are not limited to, SEQ ID NOs:24 and 25. Proteinscontaining a signal peptide are directed to the secretory pathway andare then translocated across the cell's membrane. The general mechanismof protein secretion is conserved among all gram-negative andgram-positive bacteria (Simonen and Palva (1993) Microbiol. Rev.57:109-137; Fekkes and Driessen (1999) Microbiol. Rev. 63:161-173). Allbacterial signal peptides contain a string of 13 to 20 hydrophobic aminoacids (Bae and Schneewind, J. Bacteriol., 185:2910-2919 (2003)).

[0052] Native E. coli does not hydrolyze α(1,6)-glycosidic linkages,thus the compounds containing (1,6)-linkages are not utilized infermentations. The (1,6)-linkages are hydrolyzed by both “isoamylase”and “glucosidase” enzymes (isomaltose and panose are model compounds for(1,6)-linked oligosaccharides). A recombinant E. coli containing anon-native extracellular “isoamylase” or “glucosidase” will utilizecompounds containing (1,6)-linkages (e.g., isomaltose and panose) infermentations to produce useful products. Further, any recombinantorganism containing a non-native extracellular “isoamylase” or“glucosidase” will utilize compounds containing (1,6)-linkages moreefficiently. Increased utilization efficiency will be throughconstitutive expression or altered timing of the recombinant“isoamylase” or “glucosidase” genes. Recombinant gene expression willalso increase the level of activity over that of any endogenous“isoamylase” or “glucosidase” genes that may be present, thus increasingutilization of (1,6)-linked substrate.

[0053] The present invention may be used to produce various products ofbiofermentation including, but not limited to, organic acids,antibiotics, amino acids, enzymes, vitamins, alcohols such asbioethanol, and cell mass. The bio-production of glycerol,1,3-propanediol, and cell mass using limit dextrin made available as acarbon source to the host microorganism through use of the signalpeptide serve to exemplify the invention.

[0054] The polyol, 1,3-propanediol, is a monomer useful for producingpolyester fibers and manufacturing polyurethanes and cyclic compounds. Aprocess for the biological production of 1,3-propanediol by a singleorganism from carbon substrate such as glucose or other sugars has beendescribed in U.S. Pat. No. 5,686,276, incorporated by reference herein.

[0055] Starch is a homopolysaccharide of glucose. It is synthesized inhigher plants as a granule containing two components, amylose andamylopectin (Vihinen and Mantsala, Crit. Rev. Biochem. Mol. Biol.,24:329-418 (1989)). Amylose, essentially a linear polysaccharide formedby α(1,4)-linked glucose residues, accounts for 15-25% of the granule(content varies with plant species). By contrast, amylopectin is highlybranched, with about 4 to 5% of the glucosidic linkages beingα(1,6)-linked glucose residues. Amylolytic enzymes that degrade starchare well studied. Metabolism of starch, by first degrading the polymerinto individual glucose residues in higher plant species, requires theinteraction of several amylolytic enzymes.

[0056] Amylolytic enzymes, acting alone, often only partially degradestarch into smaller linear or branched chains. Combinations ofamylolytic enzymes or enzyme combinations along with acid treatment maybe used to increase the depolymerization of starch.

[0057] Enzymes and enzyme combinations may degrade starch partially,resulting in smaller linear or branched chains, or completely toglucose. The α-glucosidases hydrolyze both (1,4)- and (1,6)-linkagesfound in oligosaccharides which are formed by the action of otheramylolytic enzymes such as α-amylases, β-amylases, glucoamylases,isoamylases and pullulanases, or by acid and heat treatments.

[0058] α-Glucosidases (α-D-glucoside glucohydrolase; for example, EC3.2.1.20) are distributed widely among microorganisms. They hydrolyze(1,4)- and (1,6)-linkages and liberate α-D-glucose units from thenonreducing end. Various types of these enzymes with different (andwide) substrate specificity have been found in bacterial species of thegenus Bacillus, Streptococcus, Escherichia, Pseudomonas,hyperthermophilic archaeobacteria such as Pyrococcus, Thermococcus, andThermotoga, and fungal species such as Penicillium, Tetrahymena,Saccharomyces, and Aspergillus.

[0059] The enzyme from Aspergillus niger has been intensively studiedfor many years and possesses wide substrate specificity. It hydrolyzessuch substrates such as maltose, kojibiose, nigerose, isomaltose,phenyl-α-glucoside, phenyl-α-maltoside, oligosaccharides, maltodextrin,and soluble starch. Similar properties are exhibited by α-glucosidasesfrom A. oryzae, Bacillus subtilis, and B. cereus and thehyperthermophilic archaea.

[0060] Oligo-(1,6)-glucosidase or isomaltase (dextrin6-α-D-glucanohydrolase, EC 3.2.1.10; coded for by the dexB gene) is anenzyme similar to α-glucosidase (Krasikov et al., Biochemistry (Moscow).66:332-348 (2001)). It catalyzes the hydrolysis of (1,6)-α-D-glucosidiclinkages in isomaltose and dextrins produced from starch and glycogen byα-amylase (Enzyme Nomenclature, C. Webb, ed. (1984) Academic Press, SanDiego, Calif.). The enzyme is less well distributed than theα-glucosidases, but is found in organisms such as Bacillus speciesincluding B. thermoglucosidius KP1006, B. cereus ATCC 7064, and possiblyB. amyloliquefaciens ATCC 23844 (Vihinen and Mantsala, Critical Reviewsin Biochemistry. 24:329-418 (1989)), as well as Bacillus coagulans(Suzuki and Tomura, Eur. J. Biochem., 158:77-83 (1986)). The Bacillusenzymes are typically 60-63 kDa in size. Anoligo-(1,6)-alpha-glucosidase (EC 3.2.1.10) has also been isolated fromThermoanaerobium Tok6-B1, with a reported molecular mass of 30-33 kDa.

[0061] The dexB enzyme from Steptococcus mutans has a pattern ofactivity similar to the dextranase enzymes (EC 3.2.1.11) that catalyzethe endohydrolysis of the (1,6)-α-D-glucosidic linkages in dextran.There is a high degree of similarity between the dexB enzyme andBacillus spp. oligo-(1,6)-glucosidases (Whiting et al., J. Gen.Microbiol., 139:2019-2026 (1993)). DexB is approximately 62 kDa in size(Aduse-Opoku et al., J. Gen Microbiol., 137:757-764 (1991)).

[0062] Enzymes with α(1,6) hydrolase activity belong to a very broadcategory of over 81 recognized families of glucosyl hydrolases(Henrissat, Biochem. J., 280:309-316 (1991); Henrissat and Bairoch,Biochem. J., 293:781-788 (1993)). The broad grouping of enzymes capableof utilizing α(1,6) linked glucose units as a fermentable substrate isfurther emphasized by demonstrating the utility of this invention, usingenzymes with as little as 69% amino acid sequence identity. Enzymes withthe ability to depolymerize oligosaccharides containing α(1,6)-linkedglucose residues are known and include glucoamylase, (EC 3.2.1.3, alsoknown as amyloglucosidase), which rapidly hydrolyzes(1,6)-α-D-glucosidic bonds or linkages when the next linkage in sequenceis a (1,4)-α-D-glucosidic linkage; α-dextrin endo-(1.6)-α-glucanosidase(EC 3.2.1.41, also known as pullulanase), which degrades(1,6)-α-D-glucosidic linkages in pullulan, amylopectin, glycogen, andthe α- and β-amylase limit dextrins of amylopectin and glycogen; sucrase(EC 3.2.1.48), which is isolated from intestinal mucosa and has activityagainst isomaltose; isoamylase (EC 3.2.1.68), which hydrolyzes the(1,6)-α-D-glucosidic linkages in glycogen, amylopectin and their β-limitdextrins; and glucan (1,6)-α-glucosidase (EC 3.2.1.70), which hydrolyzessuccessive glucose residues from (1,6)-α-D-glucans and derivedoligosaccharides.

[0063] In the context of this disclosure, a number of terms are used.

[0064] The term “starch” refers to a homopolysaccharide composed ofD-glucose units linked by glycosidic linkages that forms the nutritionalreservoir in plants. Starch occurs in two forms, amylose andamylopectin. In amylose, D-glucose units are linked exclusively byα(1,4) glycosidic linkages. Chains composed of multiple α(1,4)glycosidic linkages are considered to be linear or unbranched. Inamylopectin, while the predominant connection is via α(1,4) glycosidiclinkages, the occasional presence of an α(1,6) glycosidic linkage formsa branch point amongst the otherwise linear sections. Amylopectincontains about one α(1,6) linkage per thirty α(1,4) linkages.

[0065] The term “monosaccharide” refers to a compound of empiricalformula (CH₂O)_(n), where n≧3, the carbon skeleton is unbranched, eachcarbon atom except one contains a hydroxyl group, and the remainingcarbon atom is an aldehyde or ketone at carbon atom 2. The term“monosaccharide” also refers to intracellular cyclic hemiacetal orhemiketal forms. The most familiar monosaccharide is D-glucose. Thecyclic form of D-glucose involves reaction of the hydroxyl group ofcarbon atom 5 with the aldehyde of carbon atom 1 to form a hemiacetal,the carbonyl carbon being referred to as the anomeric carbon.

[0066] The terms “glycosidic bond” and “glycosidic linkage” refers toacetals formed by reaction of an anomeric carbon with a hydroxyl groupof an alcohol. Reaction of the anomeric carbon of one D-glucose moleculewith the hydroxyl group on carbon atom 4 of a second D-glucose moleculeleads to a (1,4) glycosidic bond or linkage. Similarly, reaction of theanomeric carbon of one D-glucose molecule with the hydroxyl group oncarbon atom 6 of a second D-glucose molecule leads to a (1,6) glycosidicbond or linkage. One skilled in the art will recognize that theglycosidic linkages may occur in α or β configurations. Glycosidiclinkage configurations are designated by, for example, α(1,4) andα(1,6).

[0067] The term “α” refers to the conformation of the linkage beingabove the plane of the ring. In contrast, a “P” linkage refers to alinkage below the plane of the ring.

[0068] The term “oligosaccharide” refers to compounds containing betweentwo and ten monosaccharide units linked by glycosidic linkages. The term“polysaccharide” refers to compounds containing more than tenmonosaccharide units linked by glycosidic linkages and generally refersto a mixture of the larger molecular weight species. A polysaccharidecomposed of a single monomer unit is referred to by the term“homopolysaccharide”.

[0069] The term “isomaltosaccharide” refers to an oligosaccharide withat least one α(1,6)-linkage.

[0070] The term “(1,4) linkage” refers to the relationship of twosaccharides in that the C1 from one saccharide unit is bonded to the C4of the second saccharide unit.

[0071] The term “(1,6) linkage” refers to the relationship of twosaccharides in that the C1 from one saccharide unit is bonded to the C6of the second saccharide unit.

[0072] The terms “amylase” and “α-amylase” refer to an enzyme thatcatalyzes the hydrolysis of an α(1,4) glycosidic linkage. The activity,hydrolysis of an α(1,4) glycosidic linkage, is referred to by the terms“amylase activity” or “amylolytic activity”. Amylases include but arenot limited to the group comprising IUBMB classifications EC 3.2.1.1(amylase), EC 3.2.1.60 ((1,4)-α-maltotetraohydrolase), and EC 3.2.1.98((1,4)-α-maltohexaosidase).

[0073] The terms “isoamylase” and “α-isoamylase” refer to an enzyme thatcatalyzes the hydrolysis of an α(1,6) glycosidic linkage. The activity,hydrolysis of an α(1,6) glycosidic linkage, is referred to by the terms“isoamylase activity” or “isoamylolytic activity”. Isoamylases includebut are not limited to the group comprising IUBMB classifications EC3.2.1.10 (oligo-(1,6)-glucosidase), EC 3.2.1.11 (dextranase), EC3.2.1.41 (pullulanase), and EC 3.2.1.68 (isoamylase).

[0074] The terms “glucosidase” and “α-glucosidase” refer to an enzymethat catalyzes the hydrolysis of both an α(1,4) glycosidic linkage andan α(1,6) glycosidic linkage and liberates α-D-glucose units from thenon-reducing end of oligosaccharides. A glucosidase has both amylolyticactivity and isoamylolytic activity. Glucosidases include but are notlimited to the group comprising IUBMB classification EC 3 2.1.3(amyloglucosidase) and EC 3.2.1.20 (α-Glucosidases).

[0075] The term “α(1,6)-linked glucose oligosaccharide hydrolyzingenzyme” refers to an enzyme possessing the functional activity tocatalyze the hydrolysis of an α(1,6) glycosidic linkage. Specificexamples of an enzyme possessing such a functional activity includeisoamylases, α-isoamylases, glucosidases, and α-glucosidases.

[0076] The term “isomaltase” or “oligo-(1,6)-glucosidase” or “dextrin6-α-D-glucanohydrolase” refers to an enzyme (EC 3.2.1.10) thathydrolyzes only α(1,6)-linkages at the nonreducing end ofoligosaccharides.

[0077] The term “DexB” refers to the (1,6)-α-glucosidase encoded by thedexB gene (GenBank Accession number M77351) of Streptococcus mutans,which releases glucose from the non-reducing ends of α(1,6)-linkedisomaltosaccharides and dextran.

[0078] The term “limit dextrin” refers to the product of the amylolyticdegradation of starch comprising monosaccharides and oligosaccharides.The action of amylase on amylopectin yields a mixture of monosaccharide(D-glucose), disaccharides (maltose, α(1,4) linked, and isomaltose,α(1,6) linked) and higher oligosaccharides. The higher oligosaccharidesmay be linear (contain exclusively α(1,4) linkages) or branched (containpredominantly α(1,4) linkages and α(1,6) linkages).

[0079] The term “degree of polymerization” or “DP” refers to the numberof monomer units present in an individual component of a saccharidemixture; for example, a monosaccharide such as D-glucose has a DP of 1,a disaccharide such as maltose has a DP of 2, a trisaccharide such aspanose has a DP of 3, etc. When applied to polysaccharide mixtures oroligosaccharide mixtures, DP refers to the average number of monomersper molecule.

[0080] The term “dextrose equivalent” (“DE”) refers to the “reducingsugar content expressed as dextrose percentage on dry matter” asdetermined by the Lane-Eynon titration. (Handbook of Starch HydrolysisProducts and their Derivatives, M. W. Kearsely and S. Z. Dziedzic, eds.,Blackie Academic & Professional, page 86). The DE scale indicates thedegree of hydrolysis of starch, starch having a nominal value of 0 DEand the ultimate hydrolysis product having a value of 100 DE.

[0081] Amylase and isoamylase activity may be intracellular orextracellular. For the purposes of this invention, the term“intracellular activity” is meant to refer to enzymatic activity thatcan be observed with disrupted cells or cell extracts when providedsubstrate but not with intact cells when provided substrateextracellularly. The term “extracellular activity” is meant to refer toactivity that is observed with intact cells (including growing cells)when provided substrate extracellularly. The inability of the enzymesubstrates to passively diffuse or be actively transported into the cellis implied in the terms “intracellular activity” and “extracellularactivity”

[0082] “Target molecule” refers to a biocatalytically-produced product.This may be a compound that is naturally produced by the biocatalyst ornon-native genes may be genetically engineered into a microorganism fortheir functional expression in the biofermentation. “Target molecule” inthis context also refers to any by-product of the biofermentation thatwould be desirable to selectively remove from the biofermentation systemto eliminate feedback inhibition and/or to maximize biocatalystactivity.

[0083] “Volumetric productivity” refers to the mass of target moleculeproduced in a biofermentor in a given volume per time, with units ofgrams/(liter hour) (abbreviated g/(L hr)). This measure is determined bythe specific activity of the biocatalyst and the concentration of thebiocatalyst. It is calculated from the titer, run time, and the workingvolume of the biofermentor.

[0084] “Titer” refers to the target molecule concentration with units ofgrams/liter (abbreviated g/L).

[0085] The terms “polynucleotide” or “polynucleotide sequence”,“oligonucleotide”, “nucleic acid sequence”, and “nucleic acid fragment”or “isolated nucleic acid fragment” are used interchangeably herein.These terms encompass nucleotide sequences and the like. Apolynucleotide may be a polymer of RNA or DNA that is single- ordouble-stranded, that optionally contains synthetic, non-natural oraltered nucleotide bases. A polynucleotide in the form of a polymer ofDNA may be comprised of one or more segments of cDNA, genomic DNA,synthetic DNA, or mixtures thereof.

[0086] The term “isolated” refers to materials, such as nucleic acidmolecules and/or proteins, which are substantially free or otherwiseremoved from components that normally accompany or interact with thematerials in a naturally occurring environment. Isolated polynucleotidesmay be purified from a host cell in which they naturally occur.Conventional nucleic acid purification methods known to skilled artisansmay be used to obtain isolated polynucleotides. The term also embracesrecombinant polynucleotides and chemically synthesized polynucleotides.

[0087] As used herein, an “isolated nucleic acid molecule” or “isolatednucleic acid fragment” is a polymer of RNA or DNA that is single- ordouble-stranded, optionally containing synthetic, non-natural or alterednucleotide bases. An isolated nucleic acid fragment in the form of apolymer of DNA may be comprised of one or more segments of cDNA, genomicDNA or synthetic DNA.

[0088] The term “complementary” is used to describe the relationshipbetween nucleotide bases that are capable to hybridizing to one another.For example, with respect to DNA, adenosine is complementary to thymineand cytosine is complementary to guanine. Accordingly, the instantinvention also includes isolated nucleic acid fragments that arecomplementary to the complete sequences as reported in the accompanyingSequence Listing as well as those substantially similar nucleic acidsequences.

[0089] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. It is therefore understood that the invention encompasses morethan the specific exemplary nucleotide or amino acid sequences andincludes functional equivalents thereof. The terms “substantiallysimilar” and “corresponding substantially” are used interchangeablyherein.

[0090] Moreover, alterations in a nucleic acid fragment that result inthe production of a chemically equivalent amino acid at a given site,but do not effect the functional properties of the encoded polypeptide,are well known in the art. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine, can also beexpected to produce a functionally equivalent product. Nucleotidechanges that result in alteration of the N-terminal and C-terminalportions of the polypeptide molecule would also not be expected to alterthe activity of the polypeptide. Each of the proposed modifications iswell within the routine skill in the art, as is determination ofretention of biological activity of the encoded products.

[0091] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6×SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65°C.

[0092] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent identity of the aminoacid sequences that they encode to the amino acid sequences disclosedherein, as determined by algorithms commonly employed by those skilledin this art. Suitable nucleic acid fragments (isolated polynucleotidesof the present invention) encode polypeptides that are at least 70%identical, preferably at least 80% identical to the amino acid sequencesreported herein. Preferred nucleic acid fragments encode amino acidsequences that are at least 85% identical to the amino acid sequencesreported herein. More preferred nucleic acid fragments encode amino acidsequences that are at least 90% identical to the amino acid sequencesreported herein. Most preferred are nucleic acid fragments that encodeamino acid sequences that are at least 95% identical to the amino acidsequences reported herein. Suitable nucleic acid fragments not only havethe above identities but typically encode a polypeptide having at least50 amino acids, preferably at least 100 amino acids, more preferably atleast 150 amino acids, still more preferably at least 200 amino acids,and most preferably at least 250 amino acids.

[0093] It is well understood by one skilled in the art that many levelsof sequence identity are useful in identifying related polypeptidesequences. Useful examples of percent identities are 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to100%. The term “% identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputinq: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5.

[0094] Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least about 60%identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are about 85% identical to the amino acid sequencesreported herein. More preferred nucleic acid fragments encode amino acidsequences that are at least about 90% identical to the amino acidsequences reported herein. Most preferred are nucleic acid fragmentsthat encode amino acid sequences that are at least about 95% identicalto the amino acid sequences reported herein.

[0095] A “substantial portion” of amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also theexplanation of the BLAST alogarithm on the world wide web site for theNational Center for Biotechnology Information at the National Library ofMedicine of the National Institutes of Health). In general, a sequenceof ten or more contiguous amino acids or thirty or more contiguousnucleotides is necessary in order to putatively identify a polypeptideor nucleic acid sequence as homologous to a known protein or gene.Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 30 or more contiguous nucleotides maybe used in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12 or more nucleotides may be used as amplificationprimers in PCR in order to obtain a particular nucleic acid fragmentcomprising the primers. Accordingly, a “substantial portion” of anucleotide sequence comprises a nucleotide sequence that will affordspecific identification and/or isolation of a nucleic acid fragmentcomprising the sequence. The instant specification teaches amino acidand nucleotide sequences encoding polypeptides that comprise one or moreparticular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention comprises the completesequences as reported in the accompanying Sequence Listing, as well assubstantial portions of those sequences as defined above.

[0096] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0097] “Synthetic nucleic acid fragments” or “synthetic genes” can beassembled from oligonucleotide building blocks that are chemicallysynthesized using procedures known to those skilled in the art. Thesebuilding blocks are ligated and annealed to form larger nucleic acidfragments which may then be enzymatically assembled to construct theentire desired nucleic acid fragment. “Chemically synthesized”, asrelated to a nucleic acid fragment, means that the component nucleotideswere assembled in vitro. Manual chemical synthesis of nucleic acidfragments may be accomplished using well-established procedures, orautomated chemical synthesis can be performed using one of a number ofcommercially available machines. Accordingly, the nucleic acid fragmentscan be tailored for optimal gene expression based on optimization of thenucleotide sequence to reflect the codon bias of the host cell. Theskilled artisan appreciates the likelihood of successful gene expressionif codon usage is biased towards those codons favored by the host.Determination of preferred codons can be based on a survey of genesderived from the host cell where sequence information is available.

[0098] The term “sequence analysis software” refers to any computeralgorithm or software program that is useful for the analysis ofnucleotide or amino acid sequences. “Sequence analysis software” may becommercially available or independently developed. Typical sequenceanalysis software will include but is not limited to the GCG suite ofprograms (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison,Wis. 53715 USA), and the FASTA program incorporating the Smith-Watermanalgorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int.Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor.Publisher: Plenum, New York, N.Y.). Within the context of thisapplication it will be understood that where sequence analysis softwareis used for analysis, that the results of the analysis will be based onthe “default values” of the program referenced, unless otherwisespecified. As used herein “default values” will mean any set of valuesor parameters that originally load with the software when firstinitialized.

[0099] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A “chimeric protein” is a protein encoded by a chimeric gene.“Endogenous gene” refers to a native gene in its natural location in thegenome of an organism. A “foreign-gene” refers to a gene not normallyfound in the host organism, but that is introduced into the hostorganism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, recombinant DNA constructs, orchimeric genes. A “transgene” is a gene that has been introduced intothe genome by a transformation procedure.

[0100] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence.

[0101] “Regulatory sequences” and “suitable regulatory sequence” referto nucleotide sequences located upstream (5′ non-coding sequences),within, or downstream (3′ non-coding sequences) of a coding sequence,and which influence the transcription, RNA processing or stability, ortranslation of the associated coding sequence. Regulatory sequences mayinclude promoters, translation leader sequences, introns, andpolyadenylation recognition sequences.

[0102] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. Promoters may bederived in their entirety from a native gene, or may be composed ofdifferent elements derived from different promoters found in nature, ormay even comprise synthetic nucleotide segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. Promoters that cause a nucleic acid fragment to be expressedin most cell types at most times are commonly referred to as“constitutive promoters”. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, nucleic acid fragments of different lengths may haveidentical promoter activity.

[0103] Promoters which are useful to drive expression of the genes ofthe present invention in a desired host cell are numerous and familiarto those skilled in the art. Virtually any promoter capable of drivingthese genes is suitable for the present invention including but notlimited to: CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1,URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1(useful for expression in Pichia); and lac, ara, tet, trp, IP_(L),IP_(R), T7, tac, and trc (useful for expression in Escherichia coli),Streptomyces lividins GI, as well as the amy, apr, and npr promoters andvarious phage promoters useful for expression in Bacillus.

[0104] “Translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

[0105] “3′ non-coding sequences” refer to DNA sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. ((1989) PlantCell 1:671-680).

[0106] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

[0107] The term “operably linked” refers to two or more nucleic acidfragments located on a single polynucleotide and associated with eachother so that the function of one affects the function of the other. Forexample, a promoter is operably linked with a coding sequence when it iscapable of affecting the expression of that coding sequence (i.e., thatthe coding sequence is under the transcriptional control of thepromoter). Coding sequences can be operably linked to regulatorysequences in sense or antisense orientation.

[0108] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0109] A “protein” or “polypeptide” is a chain of amino acids arrangedin a specific order determined by the coding sequence in apolynucleotide encoding the polypeptide. Each protein or polypeptide hasa unique function.

[0110] “Signal sequence” refers to a nucleotide sequence that encodes asignal peptide.

[0111] “Transformation” refers to the transfer of a nucleic acidfragment into a host organism or the genome of a host organism,resulting in genetically stable inheritance. Host organisms containingthe transformed nucleic acid fragments are referred to as “recombinant”,“transgenic” or “transformed” organisms. Thus, isolated polynucleotidesof the present invention can be incorporated into recombinantconstructs, typically DNA constructs, capable of introduction into andreplication in a host cell. Such a construct can be a vector thatincludes a replication system and sequences that are capable oftranscription and translation of a polypeptide-encoding sequence in agiven host cell. Typically, expression vectors include, for example, oneor more cloned genes under the transcriptional control of 5′ and 3′regulatory sequences and a selectable marker. Such vectors also cancontain a promoter regulatory region (e.g., a regulatory regioncontrolling inducible or constitutive, environmentally- ordevelopmentally-regulated, or location-specific expression), atranscription initiation start site, a ribosome binding site, atranscription termination site, and/or a polyadenylation signal.

[0112] The terms “host cell” or “host organism” refer to a microorganismcapable of receiving foreign or heterologous genes or multiple copies ofendogenous genes and of expressing those genes to produce an active geneproduct.

[0113] The terms “DNA construct” or “construct” refer to an artificiallyconstructed fragment of DNA. Such construct may be used by alone or maybe used in conjunction with a vector.

[0114] The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements, in addition to theforeign gene, that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

[0115] The terms “encoding” and “coding” refer to the process by which agene, through the mechanisms of transcription and translation, producesan amino acid sequence. The process of encoding a specific amino acidsequence includes DNA sequences that may involve base changes that donot cause a change in the encoded amino acid, or which involve basechanges which may alter one or more amino acids, but do not affect thefunctional properties of the protein encoded by the DNA sequence. It istherefore understood that the invention encompasses more than thespecific exemplary sequences.

[0116] “PCR” or “polymerase chain reaction” is well known by thoseskilled in the art as a technique used for the amplification of specificDNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

[0117] “ORF” or “open reading frame” is a sequence of nucleotides in aDNA molecule that encodes a peptide or protein. This term is often usedwhen, after the sequence of a DNA fragment has been determined, thefunction of the encoded protein is not known.

[0118] The term “fermentable carbon substrate” refers to a carbon sourcecapable of being metabolized by host organisms of the present inventionand particularly those carbon sources selected from the group consistingof monosaccharides, oligosaccharides, polysaccharides, and one-carbonsubstrates or mixtures thereof.

[0119] Isolation of Homologs

[0120] The nucleic acid fragments of the instant invention may be usedto isolate genes encoding homologous proteins from the same or othermicrobial species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No.4,683,202), ligase chain reaction (LCR), Tabor et al., Proc. Acad. Sci.USA 82, 1074, (1985)), or strand displacement amplification (SDA, Walkeret al., Proc. Natl. Acad. Sci. U.S.A., 89, 392, (1992)).

[0121] Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art. (Thein and Wallace, “The use of oligonucleotide asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986)pp. 33-50 IRL Press, Herndon, Va.); Rychlik, W. (1993) In White, B. A.(ed.), Methods in Molecular Biology, Vol. 15, pages 31-39, PCRProtocols: Current Methods and Applications. Humania Press, Inc.,Totowa, N.J.)

[0122] Hybridization methods are well defined. Typically the probe andsample must be mixed under conditions that will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed.

[0123] Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1 Mbuffers, such as sodium citrate, Tris-HCl, PIPES or HEPES (pH rangeabout 6-9), about 0.05 to 0.2% detergent, such as sodium dodecylsulfate,or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500kilodaltons), polyvinylpyrrolidone (about 250-500 kDal), and serumalbumin. Also included in the typical hybridization solution will beunlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmentednucleic DNA, e.g., calf thymus or salmon sperm DNA, or yeast RNA, andoptionally from about 0.5 to 2% wt./vol. glycine. Other additives mayalso be included, such as volume exclusion agents that include a varietyof polar water-soluble or swellable agents, such as polyethylene glycol,anionic polymers such as polyacrylate or polymethylacrylate, and anionicsaccharidic polymers, such as dextran sulfate.

[0124] Recombinant Expression-Microbial

[0125] The genes and gene products of the present sequences may beintroduced into microbial host cells. Preferred host cells forexpression of the instant genes and nucleic acid molecules are microbialhosts that can be found broadly within the fungal or bacterial familiesand which grow over a wide range of temperature, pH values, and solventtolerances. Large scale microbial growth and functional gene expressionmay utilize a wide range of simple or complex carbohydrates, organicacids and alcohols, saturated hydrocarbons such as methane or carbondioxide in the case of photosynthetic or chemoautotrophic hosts.However, the functional genes may be regulated, repressed or depressedby specific growth conditions, which may include the form and amount ofnitrogen, phosphorous, sulfur, oxygen, carbon or any trace micronutrientincluding small inorganic ions. In addition, the regulation offunctional genes may be achieved by the presence or absence of specificregulatory molecules that are added to the culture and are not typicallyconsidered nutrient or energy sources. Growth rate may also be animportant regulatory factor in gene expression. Examples of suitablehost strains include but are not limited to fungal or yeast species suchas Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula,or bacterial species such as member of the proteobacteria andactinomycetes as well as the specific genera Rhodococcus, Acinetobacter,Arthrobacter, Brevibacterium, Acidovorax, Bacillus, Streptomyces,Escherichia, Salmonella, Pseudomonas, and Cornyebacterium.

[0126]E. coli is particularly well suited to use as the hostmicroorganism in the instant invention fermentative processes. E. coliis not able to metabolize oligosaccharides containing an α(1,6) linkageand also has difficulty metabolizing any oligosaccharide of DP>7.

[0127] Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes to produce the any of the geneproducts of the instant sequences. These chimeric genes could then beintroduced into appropriate microorganisms via transformation techniquesto provide high-level expression of the enzymes.

[0128] Vectors or cassettes useful for the transformation of suitablehost cells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene harboring transcriptional initiation controls anda region 3′ of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell, although it is to beunderstood that such control regions need not be derived from the genesnative to the specific species chosen as a production host.

[0129] Initiation control regions or promoters, which are useful todrive expression of gene products. Termination control regions may alsobe derived from various genes native to the preferred hosts. Optionally,a termination site may be unnecessary, however, it is most preferred ifincluded.

[0130] For some applications it will be useful to direct the instantproteins to different cellular compartments. It is thus envisioned thatthe chimeric genes described above may be further supplemented byaltering the coding sequences to encode enzymes with appropriateintracellular targeting sequences such as transit sequences.

[0131] Enzymes Having Enhanced Activity

[0132] It is contemplated that the present sequences may be used toproduce gene products having enhanced or altered activity. Variousmethods are known for mutating a native gene sequence to produce a geneproduct with altered or enhanced activity including but not limited toerror prone PCR (Melnikov et al., Nucleic Acids Research, (Feb. 15,1999) Vol. 27, No. 4, pp. 1056-1062); site directed mutagenesis (Coombset al., Proteins (1998), 259-311, 1 plate. Editor(s): Angeletti, RuthHogue. Publisher: Academic, San Diego, Calif.) and “gene shuffling”(U.S. Pat. No. 5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat. No.5,830,721; and U.S. Pat. No. 5,837,458, incorporated herein byreference).

[0133] Pathway Modulation

[0134] Knowledge of the sequence of the present genes will be useful inmanipulating the sugar metabolism pathways in any organism having such apathway. Methods of manipulating genetic pathways are common and wellknown in the art. Selected genes in a particularly pathway may beup-regulated or down-regulated by variety of methods. Additionally,competing pathways organism may be eliminated or sublimated by genedisruption and similar techniques.

[0135] Once a key genetic pathway has been identified and sequencedspecific genes may be up-regulated to increase the output of thepathway. For example, additional copies of the targeted genes may beintroduced into the host cell on multicopy plasmids such as pBR322.Alternatively the target genes may be modified so as to be under thecontrol of non-native promoters. Where it is desired that a pathwayoperate at a particular point in a cell cycle or during a fermentationrun, regulated or inducible promoters may be used to replace the nativepromoter of the target gene. Similarly, in some cases the native orendogenous promoter may be modified to increase gene expression. Forexample, endogenous promoters can be altered in vivo by mutation,deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350;Zarling et al., PCT/US93/03868).

[0136] Within the context of the present invention it may be useful tomodulate the expression of the sugar metabolism pathway by any one of anumber of well-known methods (e.g., anti-sense, radiation- orchemically-induced mutations, gene-shuffling, etc.). For example, thepresent invention provides a number of genes encoding key enzymes in thesugar metabolism pathway leading to the production of simple sugars. Theisolated genes include the α-glucosidase and isomaltase genes. Where,for example, it is desired to accumulate glucose or maltose, any of theabove methods may be employed to overexpress the α-glucosidase andisomaltase genes of the present invention. Similarly, biosyntheticgenes' accumulation of glucose or maltose may be effected by thedisruption of down stream genes such as those of the glycolytic pathwayby any one of the methods described above.

[0137] Biofermentations

[0138] The present invention is adaptable to a variety ofbiofermentation methodologies, especially those suitable for large-scaleindustrial processes. The invention may be practiced using batch,fed-batch, or continuous processes, but is preferably practiced infed-batch mode. These methods of biofermentation are common and wellknown in the art (Brock, T. D.; Biotechnology: A Textbook of IndustrialMicrobiology, 2nd ed.; Sinauer Associates: Sunderland, Mass. (1989); orDeshpande, Appl. Biochem. Biotechnol. 36:227 (1992)).

[0139] “Biofermentation system” or “biofermentation” refers to a systemthat uses a biocatalyst to catalyze a reaction between substrate(s) andproduct(s).

[0140] The Biocatalyst

[0141] The biocatalyst initiates or modifies the rate of a chemicalreaction between substrate(s) and product(s). The biocatalyst may bewhole microorganisms or in the form of isolated enzyme catalysts. Wholemicrobial cells can be used as a biocatalyst without any pretreatmentsuch as permeabilization. Alternatively, the whole cells may bepermeabilized by methods familiar to those skilled in the art (e.g.,treatment with toluene, detergents, or freeze-thawing) to improve therate of diffusion of materials into and out of the cells.

[0142] Microorganisms useful in the present invention may include, butare not limited to, bacteria (such as the enteric bacteria Escherichiaand Salmonella, for example, as well as Bacillus, Acinetobacter,Streptomyces, Methylobacter, Rhodococcus, and Pseudomonas);cyanobacteria (such as Rhodobacter and Synechocystis); yeasts (such asSaccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula,Debatyomyces, Mucor, Pichia, and Torulopsis); filamentous fungi (such asAspergillus and Arthrobotrys); and algae. For purposes of thisapplication, “microorganism” also encompasses cells from insects,animals, or plants.

[0143] Culture Conditions

[0144] Materials and methods suitable for maintenance and growth ofmicrobial cultures are well known to those in the art of microbiology orbiofermentation science art (Bailey and Ollis, Biochemical EngineeringFundamentals, 2^(nd) Edition; McGraw-Hill: NY (1986)). Considerationmust be given to appropriate media, pH, temperature, and requirementsfor aerobic, microaerobic, or anaerobic conditions, depending on thespecific requirements of the microorganism for the desired functionalgene expression.

[0145] Media and Carbon Substrates

[0146] Biofermentation media (liquid broth or solution) for use in thepresent invention must contain suitable carbon substrates, chosen inlight of the needs of the biocatalyst. Suitable substrates may include,but are not limited to, monosaccharides (such as glucose and fructose),disaccharides (such as lactose or sucrose), oligosaccharides andpolysaccharides (such as starch or cellulose or mixtures thereof, orunpurified mixtures from renewable feedstocks (such as cheese wheypermeate, cornsteep liquor, sugar beet molasses, and barley malt). Thecarbon substrate may also be one-carbon substrates (such as carbondioxide, methanol, or methane).

[0147] In addition to an appropriate carbon source, biofermentationmedia must contain suitable minerals, salts, vitamins, cofactors,buffers, and other components, known to those skilled in the art (Baileyand Ollis, Biochemical Engineering Fundamentals, 2^(nd) ed; pp 383-384and 620-622; McGraw-Hill: New York (1986)). These supplements must besuitable for the growth of the biocatalyst and promote the enzymaticpathway necessary to produce the biofermentation target product.

[0148] Finally, functional genes that express an industrially usefulproduct may be regulated, repressed, or derepressed by specific growthconditions (for example, the form and amount of nitrogen, phosphorous,sulfur, oxygen, carbon or any trace micronutrient including smallinorganic ions). The regulation of functional genes may be achieved bythe presence or absence of specific regulatory molecules (such asgratuitous inducers) that are added to the culture and are not typicallyconsidered nutrient or energy sources. Growth rate may also be animportant regulatory factor in gene expression.

EXAMPLES

[0149] The present invention is further defined in the followingExamples. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions.

[0150] The meaning of abbreviations is as follows: “h” means hour(s),“min” means minute(s), “sec” means second(s), “d” means day(s), “mL”means milliliter(s), “L” means liter(s), “mM” means millimolar, “nm”means nanometer, “g” means gram(s), and “kg” means kilogram(s), “HPLC”means high performance liquid chromatography, “RI” means refractiveindex.

[0151] General Methods:

[0152] Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology; Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds., American Society for Microbiology: Washington,D.C. (1994) or in Biotechnology: A Textbook of Industrial Microbiology;Brock, T. D., 2^(nd) ed.; Sinauer Associates: Sunderland, Mass. (1989).

[0153] The conversion of glycerol to 1,3-propanediol was monitored byHPLC. Analyses were performed using standard techniques and materialsavailable to one of skill in the art of chromatography. One suitablemethod utilized a Waters Maxima 820 HPLC system using UV (210 nm) and RIdetection. Samples were injected onto a Shodex SH-1011 column (8 mm×300mm, purchased from Waters, Milford, Mass.) equipped with a ShodexSH-1011P precolumn (6 mm×50 mm), temperature controlled at 50° C., using0.01 N H₂SO₄ as mobile phase at a flow rate of 0.5 mL/min. Whenquantitative analysis was desired, samples were prepared with a knownamount of trimethylacetic acid as external standard. Typically, theretention times of glucose (RI detection), glycerol, 1,3-propanediol (RIdetection), and trimethylacetic acid (UV and RI detection) were 15.27min, 20.67 min, 26.08 min, and 35.03 min, respectively.

Example 1 Genome Sequencing of Bifidobacterium breve ATCC 15700

[0154]Bifidobacterium breve (ATCC 15700) was purchased from the AmericanType Culture Collection, P.O. Box 1549, Manassas, Va. 20108, U.S.A. Acell pellet was obtained and suspended in a solution containing 10 mMNa-EDTA and 50 mM Tris-HCl, pH 7.5. Genomic DNA was isolated fromBifidobacterium breve (ATCC 15700) according to standard protocols.Genomic DNA and library construction were prepared according topublished protocols (Fraser et al., Science 270 (5235):397-403 (1995)).

[0155] Genomic DNA preparation: After suspension, the cells were gentlylysed in 0.2% sarcosine, 20 mM beta-mercaptoethanol, and 150 units/mL ofLyticase and incubated for 30 min at 37° C. DNA was extracted twice withTris-equilibrated phenol and twice with chloroform. DNA was precipitatedin 70% ethanol and suspended in a solution containing 1 mM Na-EDTA and10 mM Tris-HCl, pH 7.5. The DNA solution was treated with a mix ofRNAases, then extracted twice with Tris-equilibrated phenol and twicewith chloroform. This was followed by precipitation in ethanol andsuspension in 1 mM Na-EDTA and 10 mM Tris-HCl, pH 7.5.

[0156] Library construction: 50 to 100 μg of chromosomal DNA wassuspended in a solution containing 30% glycerol, 300 mM sodium acetate,1 mM Na-EDTA, and 10 mM Tris-HCl, pH 7.5 and sheared at 12 psi for 60sec in an Aeromist Downdraft Nebulizer chamber (IBI Medical products,Chicago, Ill.). The DNA was precipitated, suspended and treated withBAL-31 nuclease. After size fractionation on a low melt agarose gel, afraction (2.0 kb or 5.0 kb) was excised, cleaned, and ligated to thephosphatased SmaI site of pUC18 (Amersham Biosciences) using T4 DNAligase (New England Biolabs, Inc., Beverly, Mass.). The ligation mix wasrun on a gel and the DNA band representing the vector plus one insertligation product was excised, treated with T4 DNA polymerase (NewEngland Biolabs), and then religated. This two-step ligation procedurewas applied to produce a high titer library with greater than 99% singleinserts.

[0157] Sequencing: A shotgun sequencing strategy approach was adoptedfor the sequencing of the whole microbial genome (Fleischmann, R. etal., Science 269(5223):496-512 (1995)). Sequence was generated on an ABIAutomatic sequencer (Applied Biosystems, Foster City, Calif.) using dyeterminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using acombination of vector and insert-specific primers. Sequence editing wasperformed in either DNAStar (DNA Star Inc., Madison, Wis.) or theWisconsin GCG program (Wisconsin Package Version 9.0, Genetics ComputerGroup (GCG), Madison, Wis.) and the CONSED package (version 7.0). Allsequences represent coverage at least two times in both directions.Sequence assembly was performed using the Phred/Phrap software package(version 0.961028.m/0.990319).

Example 2 Identification of Carbohydrate Degradation Genes

[0158] Genes encoding isoamylase activity were identified by conductingBLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol.215:403-410 (1993); see also www.ncbi.nlm.nih.gov/BLAST/) searches forsimilarity to sequences contained in the BLAST “nr” database (comprisingall non-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the SWISS-PROTprotein sequence database, EMBL, and DDBJ databases). The sequencesobtained were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTP algorithm (Gish and States, NatureGenetics 3:266-272 (1993)) provided by the NCBI.

[0159] All comparisons were done using either the BLASTN or BLASTPalgorithm. The results of the BLAST comparison are presented in Table 1,which summarizes the sequences to which they have the most similarity.Table 1 displays data based on the BLASTP algorithm with values reportedin expectation values. The expectation value (E-value) is the number ofdifferent alignments with scores equivalent to or better than aparticular score S that are expected to occur in a database search bychance. The lower the E-value, the more significant the score. TABLE 1 %% Clone Name Similarity Identified SEQ ID SEQ ID Identity^(a)Similarity^(b) E-value^(c) Citation mbc1g.pk007.h12 (AF411186) alpha- 12 61 70 0.0 Van den Broek, L. A. M. et al. Bifidobacterium breveglucosidase [Bifidobacterium “Cloning and characterization ofadolescentis] two alpha-glucosidases from Bifidobacterium adolescentis”NCBI database Mbc2g.pk018.j20 (AF358444) alpha- 3 4 73 84 0.0 Van denBroek, L. A. M. et al. Bifidobacterium breve glucosidase[Bifidobacterium “Cloning and characterization of adolescentis] twoalpha-glucosidases from Bifidobacterium adolescentis” NCBI databasembc1g.pk026.k1 (AF358444) alpha- 5 6 69 82 0.0 Van den Broek, L. A. M.et al. Bifidobacterium breve glucosidase [Bifidobacterium “Cloning andcharacterization of adolescentis] two alpha-glucosidases fromBifidobacterium adolescentis” NCBI database DexB (M77351) dextran 16 17100 100 0.0 Russell, R. R. and Ferretti, J. J. Steptococcus mutansglucosidase [Streptococcus “Nucleotide sequence of the mutans] dextranglucosidase (dexB) gene of Streptococcus mutans” J. Gen. Microbiol. 136(Pt 5), 803-810 (1990)

Example 3 Intracellular Isoamylase Activity in E. coli Containing theStreptococcus mutans dexB Gene

[0160] For cloning of the dexB gene, genomic DNA was isolated fromStreptococcus mutans (ATCC 25175D) using the protocol described inJagusztyn et al. (J. Gen. Microbiol. 128:1135-1145(1982)).

[0161] Oligonucleotide primers (SEQ ID NO:7 and SEQ ID NO:8) weredesigned based on Streptococcus mutans (dexB) DNA sequence (Ferretti etal., Infection and Immunity 56:1585-1588 (1988)) and also included BamHIand SalI restriction sites. The dexB gene was amplified using thestandard PCR protocol included with the HotStartTaq™ kit (Qiagen,Valencia, Calif.). Reactions contained 1 ng of genomic DNA and 1 μM eachof primers. The resulting 1.6 kb DNA fragment was digested with theenzymes BamHI and SalI. The digested fragment was cloned directly intothe plasmid pTRC99a (amp^(R)) (Amersham-Pharmacia, Amersham, UK)resulting in a translational fusion with the LacZ gene. The plasmid,designated pTRC99-dexB, also contains the coding sequence for the first10 amino acids of the LacZ gene, which upon expression are fused to theN-terminal end of native DexB protein. pTRC99-dexB plasmid wastransformed into E. coli DH5α cells using the manufacturer's protocol(Invitrogen, Carlsbad, Calif.) and plated on Luria Broth (LB) mediumcontaining 100 μg/mL ampicillin.

[0162] Isoamylase activity was assessed from crude protein extractfollowing expression in E. coli. A single colony of E. coliDH5α/pTRC99-dexB was cultured overnight in LB medium and then diluted1:100 into fresh LB medium (3.0 mL) and cultured for an additional twohr at 37° C. Following this incubation, the DexB gene was induced byadding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a finalconcentration of 1 mM. Crude protein was extracted from induced cellsfollowing an additional two hr incubation. To isolate the crude proteinextract, cells were collected by centrifugation (1×8000 g) and thensuspended in 0.5 mL of phosphate buffer (10 mM, pH 6.8). The suspensionwas sonicated to release total cellular protein and centrifuged(1×14,000 g) to remove cell debris. Total protein present in thesupernatant was assayed for isoamylase activity by incubation withisomaltose or separately with panose at 37° C. in 10 mM phosphate buffer(pH 6.8) for two hrs. Products of the reaction were characterized byHigh Performance Anion Exchange Chromatography (HPAEC).

[0163] For HPAEC, samples were prepared and analyzed in the followingmanner. After the two-hr incubation with isomaltose or panose, totalprotein extracts were filtered through a 0.22 μM Spin-X (R) centrifugetube filter (Costar, Corning, N.Y.) and diluted with sterile filteredwater. Samples were analyzed by HPAEC (Dionex, Sunnyvale, Calif.) usinga PA10 column, 100 mM sodium hydroxide as the eluent and a 0-150 mMsodium acetate linear gradient. Results demonstrating degradation ofisomaltose using pTRC99-dexB cell-extract are listed in Table 2.Degradation of panose, and the products formed by incubation withpTRC99-dexB cell-extract are listed in Table 3. TABLE 2 Activity of DexBCrude Protein Extract with Isomaltose (250 μg/mL) Isomaltose Cell Line(μg/mL) DH5α/pTRC99a (negative control) 256 DH5α/pTRC99-dexB ND

[0164] TABLE 3 Activity of DexB Crude Protein Extracts with Panose (150μg/mL) Panose Maltose Isomaltose Glucose Cell Line (μg/mL) (μg/mL)(μg/mL) (μg/Ml) DH5α/pTRC99a 122 ND ND ND (negative control)DH5α/pTRC99-dexB ND 74 8 82

Example 4 Expression of the Bifidobacterium breve Isoamylolytic Genes inE. coli

[0165] Several open reading frames from the Bifidobacterium breve (ATCC15700) library were identified as putative candidate genes with activityagainst α(1,6)-linked glucose oliogosaccharides (Example 2). Threeputative clones, mbc1g.pk007.h12 (h12), mbc1g.pk026.k1 (k1), andmbc2g.pk018.j20 (j20) were chosen for detailed characterization ofisoamylolytic activity, using oliogosaccharides containing α(1,6)-linkedglucose.

[0166]E. coli DH5α strains containing the cloned full length codingsequence of the putative isoamylolytic Bifidobacterium genes in pUC18from Example 1 were inoculated to LB medium and cultured at 37° C. Theculture was diluted after 20 hr (1:100) in fresh LB medium and incubatedfor an additional 3-4 hr at 37° C. Total protein extract was preparedfrom cells as described in Example 3. Total protein present in thesupernatant was assayed for isoamylolytic activity by incubation withisomaltose or separately with panose at 37° C. in 10 mM phosphate buffer(pH 6.8) for two hr. Samples were prepared and products of the reactionwere characterized by High Performance Anion Exchange Chromatography(HPAEC) as described in Example 3. Results demonstrated that the enzymesproduced from clones h12, k1, and j20 degraded isomaltose to glucose(Table 4). TABLE 4 Activity of B. breve crude extracts with Isomaltose(150 μg/mL) Isomaltose Glucose Cell line (μg/mL) (μg/mL) DH5α/pUC18 10737 (negative control) DH5α - h12 6 187 DH5α - k1 5 165 DH5α - j20 8 154

[0167] Total protein extracts were incubated with panose (250 μg/mL) fortwo hr and then filtered through a 0.22 μM Spin-X (R) centrifuge tubefilter (Costar, Corning, N.Y.). Samples were analyzed by HPAEC asdescribed in Example 3. The absence of panose following incubationdemonstrated that the enzymes produced from the clones h12, k1 and j20are capable of degrading panose. FIG. 1 shows that the clone h12degrades panose completely to glucose (also shown is the negativecontrol, plasmid pUC18 in E. coli DH5α). FIG. 1 also shows that theenzymes from the k1 and j20 clones degrade panose to glucose andmaltose.

Example 5 Expression of the Native B. breve j20 Isoamylase Gene in E.coli

[0168] The native Bifidobacterium breve gene j20 (obtained in Example 1)appeared to have a signal peptide at the NH-end of the mature codingsequence (determined by pSort prediction software; Nakai and Kanehisa,Expert, PROTEINS: Structure, Function, and Genetics 11:95-110 (1991)).The nucleic and amino acid sequences for the Bifidobacterium breve j20gene, which codes for an α(1,6)-linked glucose oligosaccharidehydrolyzing activity, are SEQ ID NO:30 SEQ ID NO:31, respectively.

[0169] Metabolism of isomaltose was, therefore, attempted using intactwhole cells. This was accomplished by culturing a single colony of E.coli DH5α cells expressing the j20 gene in LB medium containingisomaltose (500 μg/mL) at 37° C. for 24 hr. Following incubation, cellswere removed from the medium, and the medium was prepared and analyzedby HPAEC methods described in Example 3. The presence of extracellularisoamylase activity in cells expressing the B. breve j20 gene wasdemonstrated by reduced levels of isomaltose compared to the negativecontrol (E. coli DH5α cells containing only the original pUC18 plasmid).The results in Table 5 demonstrate that E. coli cells expressing thenative j20 gene degraded isomaltose supplied extracellularly. TABLE 5Isomaltose Metabolized by the Native j20 Gene Isomaltose Glucose Cellline (μg/mL) (μg/mL) DH5α/pUC18 508 26 (negative control) DH5α - j20 18022

Example 6 Extracellular Targeting of the S. mutans dexB and B. breveIsoamylase Enzymes

[0170] Because the Bifidobacterium breve k1 and Streptococcus mutansdexB genes do not appear to contain native signal peptides (pSortprediction software; Nakai and Kanehisa, Expert, PROTEINS: Structure,Function, and Genetics 11:95-110 (1991)), the mature coding sequenceswere linked in a translational fusion to signal peptides by PCR methods,allowing extracellular expression.

[0171] Modular expression vectors containing the Bacillus subtilisalkaline and neutral protease genes were constructed in a series ofsteps beginning with the plasmids pBE505 (Borchert and Nagarajan, J.Bacteriol. 173:276-282 (1991)) and pBE311 (Nagarajan and Borchert, Res.Microbiol. 142:787-792 (1991)). The plasmids were digested with therestriction enzymes KpnI and NruI. The resulting 969 bp KpnI-NruIfragment from pBE505 was isolated and ligated into the large 7.2 kbKpnI-NruI fragment from pBE311, resulting in pBE559.

[0172] Plasmids pBE559 and pBE597 (Chen and Nagarajan, J. Bacteriol.175:5697-5700 (1993)) were then digested with the restriction enzymesKpnI and EcoRV. The 941 bp KpnI-EcoRV fragment from pBE559 was ligatedinto the 8.9 kb KpnI-EcoRV fragment from pBE597, resulting in plasmidpBE592.

[0173] Plasmid pBE26 (Ribbe and Nagarajan, Mol. Gen. Genet. 235:333-339(1992)) was used as a template to amplify the B. amyloliquefaciensalkaline protease (apr) promoter region using PCR methods described inExample 3. The oligonucleotide primer SEQ ID NO:9 was designed andsynthesized to introduce an NheI restriction site at the alkalineprotease signal cleavage site and an EcoRV restriction site immediatelydownstream of the cleavage site. The oligonucleotide primer SEQ ID NO:10was designed to anneal to the 5′ polylinker region upstream of the aprpromoter region in pBE26. A PCR reaction was carried out using thedescribed primers and plasmid pBE26 template DNA. The resulting 1.2 kbPCR product was digested with KpnI and EcoRV and ligated into the largeKpnI-EcoRV fragment from pBE592, resulting in pBE92.

[0174] Plasmid pBE80 (Nagarajan et al., Gene 114:121-126 (1992)) wasused as a template to amplify the B. amyloliquefaciens neutral protease(npr) promoter region using PCR methods described in Example 3. Thedownstream primer SEQ ID NO:11 was designed and synthesized to introducean NheI restriction site at the neutral protease signal cleavage siteand an EcoRV restriction site immediately downstream of the cleavagesite. The primer SEQ ID NO:12 was designed to anneal to the 5′ region ofthe Npr promoter in pBE80. A PCR reaction was carried out using thedescribed primers and DNA template. The resulting 350 bp PCR product wasenzymatically digested with KpnI and EcoRV and ligated into the largeKpnI-EcoRV fragment from pBE592, resulting in pBE93.

[0175] A translational fusion of the k1 and dexB genes to signalpeptides of the Bacillus subtilis alkaline and neutral protease genes inthe vectors pBE92 and pBE93 was accomplished using oligonucleotideprimers described in Table 6. PCR amplification was performed by theprotocol described in Example 3, using genomic DNA from Bifidobacteriumbreve (ATCC 15700) or pTRC99-dexB plasmid, respectively, as a template.

[0176] Oligonucleotide primers SEQ ID NO:14 and SEQ ID NO:15, engineeredwith NheI and BamHI sites, were used to amplify a 1.8 kb k1 gene DNAfragment. Oligonucleotide primers SEQ ID NO:13 and SEQ ID NO:8,containing NheI and SalI restriction enzyme sites, resulted inamplification of a 1.6 kb dexB gene DNA fragment. The fragments weredigested with the appropriate enzymes and cloned into modular vectorspBE92 and pBE93.

[0177] The resulting plasmids (designated pBE92-dexB, pBE93-dexB,pBE92-k1, and pBE93-k1, respectively) contained the native enzyme linkedin a translational fusion to the signal peptide such that the signalpeptide cleavage site (Ala Ser Ala) was conserved. Nucleic and aminoacid sequences for the Bacillus subtilis neutral protease signal peptidelinked to the Bifidobacterium breve k1 gene are SEQ ID NOs:40 and 41,respectively. Nucleic and amino acid sequences for the Bacillus subtilisneutral protease signal peptide linked to the Streptococcus mutans dexBgene are SEQ ID NOs:42 and 43, respectively. The plasmids weretransformed into E. coli DH5α cells using the manufacturer's protocol(Invitrogen, Carlsbad, Calif.) and plated on Luria Broth (LB) mediumcontaining ampicillin (100 μg/mL).

[0178] Characterization of activity in E. coli DH5α cells containing thepBE93 (negative control), pBE93-dexB or pBE93-k1 plasmid was carried outby inoculating 3.0 mL of LB medium containing ampicillin (100 μg/mL) andisomaltose (0.250 mg/mL). The cells were grown at 37° C. for 20 hr.Following incubation, cells were removed from the medium and preparedand analyzed by methods described in Example 3. The presence ofextracellular isoamylase activity in cells containing the pBE93,pBE93-dexB or pBE93-k1 plasmid was demonstrated by reduced levels ofisomaltose compared to the negative control (E. coli DH5α cellscontaining only the original pBE92 plasmid). The results in Table 6demonstrate that the Npr-gene fusion proteins degraded isomaltosesupplied extracellularly. TABLE 6 DexB and K1 Extracellular FusionProtein Activity in E. coli DH5α cells Cell line Isomaltose (μg/mL)pBE93 (negative control) 215 pBE93-dexB (isolate 4) 117 pBE93-dexB(isolate 8) 89 pBE93-k1 (isolate 7) 76 pBE93-k1 (isolate 8) 74 pBE93-k1(isolate 9) 62

[0179]E. coli DH5α cells containing the pBE93-dexB or pBE93-k1 plasmidsdegraded isomaltose; however, cell growth in minimal media containingisomaltose as the sole carbon source is a much more stringent measure ofisoamylase activity. Therefore pBE93-dexB and pBE93-k1 plasmids weretransformed into the E. coli strain FM5. The FM5 strain, unlike DH5α,has the ability to grow in a minimal medium, containing only salts andtrace metals in addition to a carbon source (Maniatis et al. (1982)Molecular Cloning; a Laboratory Manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; Neidhardt (1987) Escherichia coli andSalmonella typhimurium, ASM Press, Washington, D.C.). Native FM5 cells,like the DH5α strain, cannot utilize isomaltose as a carbon source. Toconfirm this, FM5 cells transformed with the plasmid pBE93 wereinoculated into M9 media (Maniatis et al., supra; Neidhardt, supra)containing either glucose (1 mg/mL) or isomaltose (1 mg/mL) andincubated at 37° C. for at least 20 hr. Cell growth was observed after20 hr in flasks containing glucose, but not in flasks containingisomaltose, even after a 60 hr incubation.

[0180] In contrast to the negative control, FM5 cells containing theNpr-DexB and Npr-k1 fusion proteins (pBE93-dexB and pBE93-k1,respectively) grew well in M9 medium containing isomaltose following a20 hr incubation period. For this experiment FM5/pBE93, FM5/pBE93-dexBand FM5/pBE93-k1 strains were inoculated into 2.0 mL M9 mediumsupplemented with either glucose or isomaltose (1 mg/mL) as the solecarbon source. The results, shown in Table 7, indicated that when thedexB or k1 genes, are linked in a translational fusion to the Npr signalpeptide, are expressed in FM5 cells, isomaltose is metabolized andsupports cell growth. TABLE 7 DexB and K1 Extracellular Fusion ProteinActivity in E. coli FM5 cells Cell line Isomaltose (μg/mL) pBE93(negative control) 1091 pBE93-dexB (isolate 2) 319 pBE93-dexB (isolate15) 197 pBE93-dexB (isolate 3) 183 pBE93-k1 (isolate 5) 34 pBE93-k1(isolate 4) 20 pBE93-k1 (isolate 3) 17

Example 7 Expression of the Npr-dexB and Npr-k1 Fusion Genes in E. coliLeads to Increased Synthesis of Various Fermentation Products

[0181] The ability of production hosts to metabolize oligosaccharidescontaining α(1,6)-linked glucose residues may increase the yield of afermentation product when a mixture of sugars is supplied as the carbonsource. The ability of the Npr-dexB and Npr-k1 fusion proteins todegrade α(1,6)-linkages was tested by first transforming the plasmidspBE93-dexB and pBE93-k1 into a cell line engineered to produce glycerol.

[0182] One microgram of plasmid DNA was used to transform E. coli strainRJ8n (ATCC PTA-4216), which also contained the plasmid pSYCO101(spec^(R)) (described in U.S. patent application Ser. No. 10/420,587herein incorporated by reference), which encodes the DAR1 and GPP2 genesfrom Saccharomyces cerevisiase, and dhaB and orf operons from Klebsiellapnuemoniae. The transformed E. coli strain produces glycerol fromglucose as well as 1,3-propanediol when vitamin B12 is added. Methodsfor the production of glycerol and 1,3-propanediol from glucose aredescribed in detail in U.S. Pat. No. 6,358,716 and U.S. Pat. No.6,013,494 herein incorporated by reference. The transformed RJ8n cellswere plated on LB medium containing 50 μg/mL spectinomycin and 100 μg/mLampicillin. Single colonies were used to inoculate 2.0 mL of TM2 medium(potassium phosphate, 7.5 g/L; citric acid, 2.0 g/L; ammonium sulfate,3.0 g/L; magnesium sulfate, 2.0 g/L; calcium chloride, 0.2 g/L; ferricammonium citrate, 0.33 g/L; yeast extract (Difco-BD, Sparks, Md.) 5.0g/L; trace elements (zinc sulfate, copper sulfate, cobalt chloride,manganese sulfate, ferric sulfate, sodium chloride); ammonium hydroxide,pH to 6.5; also containing glucose or isomaltose (1 mg/mL). Cultureswere grown for 24 hr at 37° C. Cells were prepared and analyzed bymethods described in Example 3.

[0183] Glycerol was shown to accumulate when E. coli RJ8n cellscontaining only the plasmid pSYCO101 were cultured for 24 hr at 37° C.in TM2 medium with glucose as the carbon source (Table 8). However, thisnegative control line produced negligible levels of glycerol whenisomaltose was substituted for glucose in the medium, demonstrating thatα(1,6)-linked glucose does not support accumulation of a fermentationproduct. By contrast, glycerol was produced in E. coli RJ8n containingthe plasmids pSYCO101 and pBE93-dexB or pBE93-k1 when either isomaltoseor glucose was provided as sole carbon sources (Table 8). Whenisomaltose was used as a carbon source, glycerol production was shown tobe 8 to 9 times higher in E. coli RJ8n containing both the pBE93-dexBand pSYCO101 plasmids as compared to the negative control line, RJ8ncontaining only pSYCO101. Glycerol accumulation, using isomaltose, was 6to 10 times higher in lines containing pSYCO101 and pBE93-k1 as comparedto the negative control. The data in Table 8 demonstrate that expressionof the Npr-dexB or Npr-k1 genes resulted in glycerol production incultures supplied with isomaltose. The data further demonstrate thatlevels of product accumulated were comparable for cultures containingthe fusion proteins regardless of whether the carbon source was glucoseor isomaltose. TABLE 8 Glycerol Accumulation Due to Expression ofNpr-DexB or Npr-K1 Glycerol Accumulated (μg/mL) Glucose-suppliedIsomaltose- Cell line cultures supplied cultures RJ8n/pSYCO101 430 39RJ8n/pSYCO101/pBE93- 381 362 dexB (isolate 4) RJ8n/pSYCO101/pBE93- 353354 dexB (isolate 8) RJ8n/pSYCO101/pBE93- 383 401 k1 (isolate 2)RJ8n/pSYCO101/pBE93- 412 226 k1 (isolate 6)

[0184] The capability of E. coli line RJ8n containing the plasmidspSYCO101 and pBE93-k1 to produce fermentation products usingα(1,6)-linked glucose as a substrate was further characterized byculturing in TM2 medium containing panose (1 mg/mL) and comparing theresults to the same line using glucose as a substrate (1 mg/mL).

[0185] Data in Table 9 also show that E. coli strain RJ8n containingonly the plasmid pSYCO101 (negative control) does not synthesizeglycerol when panose is supplied as the sole carbohydrate source in TM2medium. However, glycerol is produced when the plasmid pBE93-k1 ispresent in this same strain and cultured in TM2 medium with panose.Glycerol accumulation in E. coli RJ8n containing the plasmids pSYCO101and pBE93-k1 was comparable when either glucose or panose was suppliedas a carbohydrate source. TABLE 9 Glycerol Accumulation Due toExpression of Npr-K1 Glycerol Accumulated (μg/mL) Glucose-suppliedIsomaltose- Cell line cultures supplied cultures RJ8n/pSYCO101 417 25RJ8n/pSYCO101/pBE93-k1 (9) 396 363 RJ8n/pSYCO101/pBE93-k1 (7) 376 347

[0186] The data above demonstrate that expression of the Npr-dexB orNpr-k1 fusion protein in E. coli results in increased production ofglycerol when isomaltose or panose represents the sole carbohydratesource in the medium. Demonstrating that this result is not limited toglycerol production alone was accomplished by synthesis of anotherfermentation product (1,3-propanediol) using the same fusion proteinexpression system.

[0187] RJ8n cells transformed with the plasmids pSYCO101 and pBE93-dexBor pBE93-k1 were used to inoculate 2.0 mL of TM2 medium (describedabove) also containing glucose (1 mg/mL) or isomaltose (1 mg/mL) andvitamin B12 (100 ng/L). Cultures were grown for 20 hr at 37° C. Cellswere prepared and analyzed by methods described in Example 3.

[0188] The data in Table 10 demonstrate that 1,3-propanediol was notsynthesized by the negative control line (RJ8n/pSYCO101) when grown inmedia containing only isomaltose as a carbohydrate source. However, wheneither the Npr-dexB or Npr-k1 fusion protein was expressed in RJ8ncells, isomaltose was shown to be metabolized. This resulted inaccumulation of the fermentation product 1,3-propanediol. The datafurther demonstrate that the level of 1,3-propanediol synthesized byRJ8n cells expressing the Npr-dexB or Npr-K1 fusion protein wascomparable whether glucose or isomaltose was supplied as the solecarbohydrate. TABLE 10 1,3-Propanediol Accumulation Due to Expression ofNpr--dexB or Npr-k1 1,3-Propanediol (mg/mL) Glucose- Isomaltose-supplied supplied Isomaltose Cell line cultures cultures (μg/mL)RJ8n/pSYCO101 2.8 ND 1225 RJ8n/pSYCO101/pBE93-k1 (9) 1.7 2.8 12RJ8n/pSYCO101/pBE93-k1 (7) 3.0 2.9 14 RJ8n/pSYCO101/pBE93-dexB 3.0 3.127

Example 8 Expression of the B. breve k1 Gene in E. coli Using anAlternative Promoter

[0189] The use of alternative promoters to direct expression of apreferred gene is often highly desirable. Alternative promoters may beused to vary the level or timing of gene expression and, therefore,increase utilization of a preferred substrate.

[0190] Effective expression of the B. breve k1 isoamylase gene using analternative promoter was demonstrated by replacing the neutral proteasepromoter in the plasmid pBE93-k1 (Example 6) with a glucose isomerase(GI) promoter and variant of the GI-promoter. Isolation of theStreptomyces lividins GI-promoter and creation of the variant promoterwas disclosed in U.S. patent application Ser. 10/420,587. Prior toreplacing the NPR-promoter, modifications of the non-coding nucleotidesequences of the neutral protease signal peptide and K1 gene were made.The sequence modifications resulted in restriction enzyme sites, whichwould be used in subsequent cloning steps.

[0191] The restriction enzyme sites SacI and PacI were added to the 5′and 3′-ends of the neutral protease signal peptide and K1 genesequences, respectively, by PCR using the primers SEQ ID NO. 18 and SEQID NO. 19. PCR amplfication was performed by the protocol described inExample 3. A 1919 bp PCR product was isolated and ligated into thepSYCO109mcs wild-type GI yqhD plasmid as disclosed in U.S. patentapplication Ser. No. 10/420,587, which was also digested with theenzymes SacI and PacI. The resulting plasmid contains a wild-type GIpromoter and the NPR-signal sequence linked in a translational fusion tothe k1 gene. This construct was designated WTGI-ss-K1. A variant GIpromoter was also used to direct expression of the NPR-signal peptide/K1fusion. A 1919 bp PCR product, resulting from a reaction using theprimers SEQ ID NO:18 and SEQ ID NO:19 was placed into thepSYCO109mcs-short 1.6 GI yqhD plasmid, using SacI and PacI restrictionenzyme sites. The resulting plasmid was designated LowGI-ss-K1. Thisvariant promoter when operably linked to a yqhD gene was previouslyshown to direct lower levels of gene expression (U.S. patent applicationSer. No. 10/420,587) as compared to the wild-type GI promoter-yqhDconstruct.

[0192] Demonstrating effective expression of the K1 gene using thewild-type and variant GI promoters was accomplished by an activityassay. E. coli cells (strain DH5α, Invitrogen, Carlsbad, Calif.) weretransformed with the plasmids WTGI-ss-K1 and LowGI-ss-K1 and grownovernight in LB medium. Cell pellets were recovered by centrifugationand suspended in {fraction (1/10)} volume sodium-phosphate buffer (10mM, pH 7.0). The cells in the suspension were lysed with a French pressand cell-debris was removed by centrifugation. Total proteinconcentration was determined by Bradford assay (Bio-Rad, Hercules,Calif.). Activity of the K1 gene product in a total protein isolate wasassayed using 4-nitrophenyl-α-D-glucopyranoside (PNPG, Sigma, ST. Louis,Mo.). Total protein extract from cells containing the plasmidsWTGI-ss-K1, LowGI-ss-K1, NPR-ss-K1 (positive control) and pSYCO109(negative control) were incubated in a10 mM sodium phosphate bufferedsolution containing 10 mM PNPG for up to 30 min at 30° C. Release of theglucose residue from PNPG results in PNP accumulation, which absorbslight at 400 nm. PNP accumulation as a direct result of k1 enzymeactivity was monitored over time by absorbance at a wavelength of 400nm. Table 11 below demonstrates that a promoter, other than the neutralprotease promoter, may be used to direct expression of an active K1gene. The results also demonstrate that an alternative promoter may beused to modify the level of K1 expression and that K1 activitycorresponds to the relative level of promoter strength. TABLE 11 Rate ofPNP production resulting from K1 enzyme activity Plasmid Activity (mMPNP/mg protein min⁻¹) WTGI-ss-K1 (high expresser) 0.0144 NPR-ss-K1(positive control) 0.0104 LowGI-ss-K1 (low expresser) 0.0028 pSYCO109(negative control) 0.0002

Example 9 Integration of the B. breve k1 Gene into the E. coli Genome

[0193] Integrating the desired DNA into the cell's genome may enhancethe stability of gene expression over time and under a variety offermentation conditions. However, the location of integration may affectgene expression level and, ultimately, the effectiveness of the desiredenzyme activity.

[0194] Integration of the k1 expression cassette (NPR promoter-signalpeptide-k1 gene) into the genome of E. coli (strain FM5) and thedemonstration of utility by the use of an α(1,6)-linked glucosesubstrate was accomplished by first cloning into the plasmid pKD3(Datsenko and Wanner, Proc. Natl. Acad. Sci. 97:6640-6645 (2000)). Thehost aldA (aldehyde dehydrogenase A) and aldB (aldehyde dehydrogenase B)genomic sites were chosen for integration. PCR primers were designedthat had homology to the plasmid pKD3, aldA or aldB and k1 genesequences (SEQ ID NOs:20 through 23).

[0195] PCR amplification was performed by the protocol described inExample 3. PCR products resulting from a reaction with the primers SEQID NOs. 21-23 and the plasmid pKD3 containing the k1 expression cassettewere isolated, ligated and transformed into E. coli (FM5). Cellscontaining the integrated k1 expression cassette were selected by growthon LB medium containing chloramphenicol. Chloramphenicol positivecolonies were tested for the presence of the k1 gene by PCR reaction,using the primers SEQ ID NO:7 and SEQ ID NO:8.

[0196] FM5 lines containing the integrated k1 expression cassette werefurther tested for activity by growth analysis in media containingisomaltose as the sole carbohydrate source. Chloramphenicol and PCRpositive colonies were inoculated into TM2 medium (see Example 7) with0.5% isomaltose (w/v) and grown at 35° C. Samples were removed atvarious time points and characterized for cell mass accumulation byoptical density (A600 nm) and isomaltose consumption (by HPLC, seeGeneral Methods).

[0197] Table 12 below demonstrates that FM5 cells alone do notmetabolize isomaltose when provided as the sole carbohydrate source.This is shown by the low level of cell mass accumulation when grown inTM2 medium with 0.5% isomaltose. Low-level growth of the negative lineFM5 was observed, but due only to a small amount of the fermentablesugar maltose contaminating the isomaltose source material (Sigma, St.Louis, Mo.). Cells containing the integrated K1 expression cassette grewat a much higher rate and to a higher final optical density followingthe 25 hr time period. A PCR-positive colony containing the k1expression cassette integrated at the aldA site was designated A2-3.Colonies, positive by PCR, containing the k1 expression cassetteintegrated at the aldB site were designated B1-1 and B1-2. TABLE 12 Cellmass accumulation (A600 nm) Time (hours) FM5 FM5-A2-3 FM5-B1-1 FM5-B1-20 0.02 0.02 0.02 0.02 3 0.66 0.72 0.76 0.75 6 2.75 3.17 6.60 6.01 8 3.344.50 10.40 9.92 11 3.72 8.34 10.41 10.10 25 3.66 10.16 11.10 10.78

[0198] Isomaltose consumption by cells containing the integrated K1expression cassette was also compared to the FM5 negative control lineby HPLC analysis. The data in Table 13 demonstrate that the K1expression cassette is active following integration and allows cells tocompletely utilize available sugar containing α(1,6)-linked glucose,compared to the negative control which does not utilize thiscarbohydrate. The data also show that isomaltose is not consumed at thesame rate in lines where the gene has been integrated into the aldA, ascompared to the aldB, sites. TABLE 13 Isomaltose Consumption (g/L) Time(hours) FM5 FM5-A2-3 FM5-B1-1 FM5-B1-2 0 5.56 5.46 5.36 5.31 3 5.52 5.355.31 5.30 6 5.60 4.73 1.81 1.78 8 5.48 3.64 0 0 11 5.77 1.34 0 0 25 5.550 0 0

[0199]

1 43 1 1815 DNA Bifidobacterium breve ATCC#15700 1 atgaccgcca acaacctcaatgacgactgg tggaagcagg ccgtcgttta ccagatttac 60 ccgcgcagct tcaaggacgttaacggcgac ggcatcggcg acatcgccgg cgttaccgag 120 aaaatggact acctgaaaaacctcggcgtg gacgccatct ggctctcccc gttctacccc 180 tccgatctgg cggacggcggctacgacgtg atcgactacc gcaacgtcga cccgcgactg 240 ggcaccatgg acgacttcgacgccatggcc aaagccgcgc atgaggccgg catcaaggtg 300 atcgtggaca tcgtgcccaatcacaccgcc gacaagcacg tgttcttcaa ggaagccctc 360 gccgccgagc ccggctccccggcgcgcgac cgctacatct tccgcgacgg ccgcggcgag 420 cacggcgaac tgccgcccaacgactggcag tccttcttcg gcggcccggc ctgggctcgc 480 gtggccgacg gccagtggtatctgcacctg ttcgacaagg cgcaaccgga cgtcaactgg 540 aagaacccgg acatccacgaggaattcaag aaaaccctgc gcttctggtc cgaccacggc 600 accgacggct tccgcatcgacgtggcgcac ggtctggcca aagaccttga atccaagccg 660 ctggaggagc tcggccgcgaatacagcgtg gtcggcgtgc tgaatcacga cttcagccat 720 ccgctgttcg accgccgcgaagtgcacgac atctaccgcg aatggcgcaa ggtgttcaac 780 gagtacgacc cgccgcgctttgccgtggcc gaggcgtggg tggtacccga gcaccagcac 840 ctgtatgcct cgatggatgagctggggcag tccttcaact tcgactttgc gcaggccagc 900 tggtatgccg atgagttccgcgcagccatc gccgcgggtc tcaaggccgc tgccgaaacc 960 ggcggttcca ccaccacgtgggtgatgaac aaccatgacg tgccgcgcag cccctcccgc 1020 tatggtctac cgcaggtcaagggcgcgcct taccaccagc tgccgcacga ctggctgctg 1080 cgcaacggca ccacgtatcccgaggatcgc gagcttggca cccgccgcgc ccgcgccgcc 1140 gctttgatgg agctcggcctgcccggcgcc gcctatatct atcagggcga ggagctgggc 1200 ctgtttgaag tggccgatattccgtgggat cgactggaag atccgaccgc tttccacacc 1260 gctcaggcca cgatggacaagggccgagac ggctgccgcg tgccgattcc gtggaccgct 1320 gcaaacgaac cgaccttggctgatttcagc cgcccgatcc cggccgatga cggcaccggc 1380 gagaaccacg tgccgctgtgcgccgccggc cagttcggca cgggcgcttc cttcggcttc 1440 tcgccggcta cgcgcgctgagggcgtgacg ccggccgccg acccgcacct gccgcagccg 1500 ttgtggttca aggattacgcggtggacgtg gagcaggccg acccggattc aatgctcgcg 1560 ctgtatcatg cggcgttggcgattcgtcag gagtcgctga ccgccacgcg tgacaccacc 1620 gctgagcagg tggatatggggccggacgtg gtggcctaca cccgcgcggc ggttggtggc 1680 cgcacgttca cctcgatcaccaacttcggc accgagccgg tggagctgcc tggaggctcc 1740 gtcgtgctga cgtccggcccgctgaccccc gacggccagc tccccaccga cacttctgcg 1800 tgggttatca agtag 1815 2604 PRT Bifidobacterium breve ATCC#15700 2 Met Thr Ala Asn Asn Leu AsnAsp Asp Trp Trp Lys Gln Ala Val Val 1 5 10 15 Tyr Gln Ile Tyr Pro ArgSer Phe Lys Asp Val Asn Gly Asp Gly Ile 20 25 30 Gly Asp Ile Ala Gly ValThr Glu Lys Met Asp Tyr Leu Lys Asn Leu 35 40 45 Gly Val Asp Ala Ile TrpLeu Ser Pro Phe Tyr Pro Ser Asp Leu Ala 50 55 60 Asp Gly Gly Tyr Asp ValIle Asp Tyr Arg Asn Val Asp Pro Arg Leu 65 70 75 80 Gly Thr Met Asp AspPhe Asp Ala Met Ala Lys Ala Ala His Glu Ala 85 90 95 Gly Ile Lys Val IleVal Asp Ile Val Pro Asn His Thr Ala Asp Lys 100 105 110 His Val Phe PheLys Glu Ala Leu Ala Ala Glu Pro Gly Ser Pro Ala 115 120 125 Arg Asp ArgTyr Ile Phe Arg Asp Gly Arg Gly Glu His Gly Glu Leu 130 135 140 Pro ProAsn Asp Trp Gln Ser Phe Phe Gly Gly Pro Ala Trp Ala Arg 145 150 155 160Val Ala Asp Gly Gln Trp Tyr Leu His Leu Phe Asp Lys Ala Gln Pro 165 170175 Asp Val Asn Trp Lys Asn Pro Asp Ile His Glu Glu Phe Lys Lys Thr 180185 190 Leu Arg Phe Trp Ser Asp His Gly Thr Asp Gly Phe Arg Ile Asp Val195 200 205 Ala His Gly Leu Ala Lys Asp Leu Glu Ser Lys Pro Leu Glu GluLeu 210 215 220 Gly Arg Glu Tyr Ser Val Val Gly Val Leu Asn His Asp PheSer His 225 230 235 240 Pro Leu Phe Asp Arg Arg Glu Val His Asp Ile TyrArg Glu Trp Arg 245 250 255 Lys Val Phe Asn Glu Tyr Asp Pro Pro Arg PheAla Val Ala Glu Ala 260 265 270 Trp Val Val Pro Glu His Gln His Leu TyrAla Ser Met Asp Glu Leu 275 280 285 Gly Gln Ser Phe Asn Phe Asp Phe AlaGln Ala Ser Trp Tyr Ala Asp 290 295 300 Glu Phe Arg Ala Ala Ile Ala AlaGly Leu Lys Ala Ala Ala Glu Thr 305 310 315 320 Gly Gly Ser Thr Thr ThrTrp Val Met Asn Asn His Asp Val Pro Arg 325 330 335 Ser Pro Ser Arg TyrGly Leu Pro Gln Val Lys Gly Ala Pro Tyr His 340 345 350 Gln Leu Pro HisAsp Trp Leu Leu Arg Asn Gly Thr Thr Tyr Pro Glu 355 360 365 Asp Arg GluLeu Gly Thr Arg Arg Ala Arg Ala Ala Ala Leu Met Glu 370 375 380 Leu GlyLeu Pro Gly Ala Ala Tyr Ile Tyr Gln Gly Glu Glu Leu Gly 385 390 395 400Leu Phe Glu Val Ala Asp Ile Pro Trp Asp Arg Leu Glu Asp Pro Thr 405 410415 Ala Phe His Thr Ala Gln Ala Thr Met Asp Lys Gly Arg Asp Gly Cys 420425 430 Arg Val Pro Ile Pro Trp Thr Ala Ala Asn Glu Pro Thr Leu Ala Asp435 440 445 Phe Ser Arg Pro Ile Pro Ala Asp Asp Gly Thr Gly Glu Asn HisVal 450 455 460 Pro Leu Cys Ala Ala Gly Gln Phe Gly Thr Gly Ala Ser PheGly Phe 465 470 475 480 Ser Pro Ala Thr Arg Ala Glu Gly Val Thr Pro AlaAla Asp Pro His 485 490 495 Leu Pro Gln Pro Leu Trp Phe Lys Asp Tyr AlaVal Asp Val Glu Gln 500 505 510 Ala Asp Pro Asp Ser Met Leu Ala Leu TyrHis Ala Ala Leu Ala Ile 515 520 525 Arg Gln Glu Ser Leu Thr Ala Thr ArgAsp Thr Thr Ala Glu Gln Val 530 535 540 Asp Met Gly Pro Asp Val Val AlaTyr Thr Arg Ala Ala Val Gly Gly 545 550 555 560 Arg Thr Phe Thr Ser IleThr Asn Phe Gly Thr Glu Pro Val Glu Leu 565 570 575 Pro Gly Gly Ser ValVal Leu Thr Ser Gly Pro Leu Thr Pro Asp Gly 580 585 590 Gln Leu Pro ThrAsp Thr Ser Ala Trp Val Ile Lys 595 600 3 1845 DNA Bifidobacterium breveATCC#15700 3 atgaataagg agccaacgat gactactttc aaccgcacaa taattcccgacgccattcgc 60 accaacggag ccacgcccaa cccgtggtgg tcgaacgccg tcgtctaccagatttaccca 120 cgttccttcc aggacacgaa cggcgatggt ctcggcgacc tgaagggcatcacctcccgc 180 ctcgactatc tcgccgacct cggcgtggat gtgctgtggc tctctccggtctacaggtcc 240 ccgcaagacg acaacggcta cgacatctcc gactaccggg acatcgacccgctgttcggc 300 acgcttgacg acatggacga gctgctcgcc gaagcgcaca agcgcggcctcaagatcgtg 360 atggacctgg tggtcaatca cacctctgac gagcacgcgt ggttcgaggcgtcgaaggac 420 aaggacgacc cgcacgccga ctggtactgg tggcgtcccg cccgccccggccacgagccg 480 ggcacgcccg gcgccgagcc gaatcagtgg ggctcctact tcggcggttccgcatgggag 540 tacagcccgg agcgcggcga gtactacctg caccagttct cgaagaagcagcctgatctc 600 aactgggaga acccggccgt gcgccgtgca gtgtacgaca tgatgaactggtggctcgat 660 cgcggcatcg acggcttccg tatggacgtc atcaccctta tctccaagcgcaccgacccc 720 aacggcaggc tccccggcga ggccggttcc gagctgcagg acctgccggtgggggaggag 780 ggctactccg acccgaatcc gttctgtgcg gacggccccc gtcaggatgaattcctggct 840 gaaatgcgcc gtgaggtatt cgaagggcgt gacggcttcc tgactgtaggcgaggcgcca 900 ggcgtcacag cccagcgcaa cgaatacatc accgatccgg ccaatggcgagctggatatg 960 ctcttcctat ttgagcatgt tgattttgat tgcgaaggta ccaagtggaagccgttgccg 1020 ctcgatctgc cgaagcttaa gagcatcatg gccggctatc aggccgctgtgcagaacgca 1080 ggatgggcca gcctattcac cggcaaccac gatcagccgc gcgtggtttcccgctggggt 1140 gacgattcct cggaagaggc tcgggtccgc tcggccaagg cccttggcctgatgctgcac 1200 ctgcaccgtg gtaccccgta catctatcag ggtgaagaat tgggcatgaccgacgcccac 1260 ttcactcgtc tcgaccagta ccgcgacctt gaatccctga acgcctaccgtcaaagggtc 1320 gaagaggcca aggtgcagtc gcccgaatcc atgatggccg gtatcgccgcccgcggtcgt 1380 gacaactcac gcacaccgat gcagtgggat ggctccgtct acgccggtttcaccgcacct 1440 gacgcagccg ccgagccatg gatctccgtg aatccgaatc atgccgagatcaacgccgcc 1500 ggcgaattcg atgatccgga ttcggtgtac tccttctaca agcggctcatcgcgctgcgc 1560 cacgacatgc ctgtcgtgga ggccggcgac tggcatctgc tcgacgcggacgatgcgcat 1620 gtgtatgcct tcactcgtac cctcggtgac gagaagttgt tggtcgtggtcaatatgtcc 1680 gggcgaactg ttgatttgcc tcgcgaatcc gccgaactgt tggcagtggccgatggcctt 1740 gccgagtcga acgtggtgat cagcacgtat gatgccccgc acgctgtgaccgctcttgcc 1800 ggccgtgagc ttgcaccatg ggagggcgtc gtcgtcagcc tataa 1845 4613 PRT Bifidobacterium breve ATCC#15700 4 Met Asn Lys Glu Pro Thr MetThr Thr Phe Asn Arg Thr Ile Ile Pro 1 5 10 15 Asp Ala Ile Arg Thr AsnGly Ala Thr Pro Asn Pro Trp Trp Ser Asn 20 25 30 Ala Val Val Tyr Gln IleTyr Pro Arg Ser Phe Gln Asp Thr Asn Gly 35 40 45 Asp Gly Leu Gly Asp LeuLys Gly Ile Thr Ser Arg Leu Asp Tyr Leu 50 55 60 Ala Asp Leu Gly Val AspVal Leu Trp Leu Ser Pro Val Tyr Arg Ser 65 70 75 80 Pro Gln Asp Asp AsnGly Tyr Asp Ile Ser Asp Tyr Arg Asp Ile Asp 85 90 95 Pro Leu Phe Gly ThrLeu Asp Asp Met Asp Glu Leu Leu Ala Glu Ala 100 105 110 His Lys Arg GlyLeu Lys Ile Val Met Asp Leu Val Val Asn His Thr 115 120 125 Ser Asp GluHis Ala Trp Phe Glu Ala Ser Lys Asp Lys Asp Asp Pro 130 135 140 His AlaAsp Trp Tyr Trp Trp Arg Pro Ala Arg Pro Gly His Glu Pro 145 150 155 160Gly Thr Pro Gly Ala Glu Pro Asn Gln Trp Gly Ser Tyr Phe Gly Gly 165 170175 Ser Ala Trp Glu Tyr Ser Pro Glu Arg Gly Glu Tyr Tyr Leu His Gln 180185 190 Phe Ser Lys Lys Gln Pro Asp Leu Asn Trp Glu Asn Pro Ala Val Arg195 200 205 Arg Ala Val Tyr Asp Met Met Asn Trp Trp Leu Asp Arg Gly IleAsp 210 215 220 Gly Phe Arg Met Asp Val Ile Thr Leu Ile Ser Lys Arg ThrAsp Pro 225 230 235 240 Asn Gly Arg Leu Pro Gly Glu Ala Gly Ser Glu LeuGln Asp Leu Pro 245 250 255 Val Gly Glu Glu Gly Tyr Ser Asp Pro Asn ProPhe Cys Ala Asp Gly 260 265 270 Pro Arg Gln Asp Glu Phe Leu Ala Glu MetArg Arg Glu Val Phe Glu 275 280 285 Gly Arg Asp Gly Phe Leu Thr Val GlyGlu Ala Pro Gly Val Thr Ala 290 295 300 Gln Arg Asn Glu Tyr Ile Thr AspPro Ala Asn Gly Glu Leu Asp Met 305 310 315 320 Leu Phe Leu Phe Glu HisVal Asp Phe Asp Cys Glu Gly Thr Lys Trp 325 330 335 Lys Pro Leu Pro LeuAsp Leu Pro Lys Leu Lys Ser Ile Met Ala Gly 340 345 350 Tyr Gln Ala AlaVal Gln Asn Ala Gly Trp Ala Ser Leu Phe Thr Gly 355 360 365 Asn His AspGln Pro Arg Val Val Ser Arg Trp Gly Asp Asp Ser Ser 370 375 380 Glu GluAla Arg Val Arg Ser Ala Lys Ala Leu Gly Leu Met Leu His 385 390 395 400Leu His Arg Gly Thr Pro Tyr Ile Tyr Gln Gly Glu Glu Leu Gly Met 405 410415 Thr Asp Ala His Phe Thr Arg Leu Asp Gln Tyr Arg Asp Leu Glu Ser 420425 430 Leu Asn Ala Tyr Arg Gln Arg Val Glu Glu Ala Lys Val Gln Ser Pro435 440 445 Glu Ser Met Met Ala Gly Ile Ala Ala Arg Gly Arg Asp Asn SerArg 450 455 460 Thr Pro Met Gln Trp Asp Gly Ser Val Tyr Ala Gly Phe ThrAla Pro 465 470 475 480 Asp Ala Ala Ala Glu Pro Trp Ile Ser Val Asn ProAsn His Ala Glu 485 490 495 Ile Asn Ala Ala Gly Glu Phe Asp Asp Pro AspSer Val Tyr Ser Phe 500 505 510 Tyr Lys Arg Leu Ile Ala Leu Arg His AspMet Pro Val Val Glu Ala 515 520 525 Gly Asp Trp His Leu Leu Asp Ala AspAsp Ala His Val Tyr Ala Phe 530 535 540 Thr Arg Thr Leu Gly Asp Glu LysLeu Leu Val Val Val Asn Met Ser 545 550 555 560 Gly Arg Thr Val Asp LeuPro Arg Glu Ser Ala Glu Leu Leu Ala Val 565 570 575 Ala Asp Gly Leu AlaGlu Ser Asn Val Val Ile Ser Thr Tyr Asp Ala 580 585 590 Pro His Ala ValThr Ala Leu Ala Gly Arg Glu Leu Ala Pro Trp Glu 595 600 605 Gly Val ValVal Ser 610 5 1818 DNA Bifidobacterium breve ATCC#15700 5 atgatgacctctttcaaccg tgaacccctg cccgacgccg tccgcacgaa tggcgcctcc 60 ccgaacccgtggtggtcgaa cgccgtcgtc taccagattt acccacgttc cttccaggac 120 acgaacggcgatggtttcgg agatcttaag ggcattactt cccgcctcga ctatcttgcc 180 gacctcggcgtggatgtgct gtggctctcc ccggtctaca ggtccccgca agacgacaac 240 ggctacgacatctccgacta ccgggacatc gacccgctgt tcggcacgct cgacgacatg 300 gacgagctgctcgccgaagc gcacaagcgc ggcctcaaga tcgtgatgga cctggtcgtc 360 aaccacacctccgacgagca cgcgtggttc gaggcgtcga aggacaagga cgacccgcac 420 gccgactggtactggtggcg tcccgcccgc cccggccacg agcccggcac gcccggcgcc 480 gagccgaaccagtggggctc ctacttcggc ggctccgcat gggaatattg ccccgagcgt 540 ggtgagtactatctccacca gttctcgaag aagcagccgg acctcaactg ggagaacccg 600 gccgtacgccgagccgtgta cgacatgatg aactggtggc tcgatcgcgg catcgacggc 660 ttccgcatggacgtcatcac cctgatctcc aagcgcacgg atgcaaacgg caggctgccc 720 ggcgagaccggttccgagct gcaggacctg ccggtggggg aggagggcta ctccaacccg 780 aacccgttctgcgccgacgg tccgcgtcag gacgagttcc tcgccgagat gcgccgcgag 840 gtgttcgacgggcgtgacgg cttcctcacc gtcggcgagg cccccggcat caccgccgaa 900 cgcaacgagcacatcaccga ccccgccaac ggcgagctgg atatgctctt cctgttcgaa 960 cacatgggcgtcgaccaaac ccccgaatcg aaatgggacg acaaaccatg gacgccggcc 1020 gacctcgaaaccaagctcgc cgaacaacag gacgccatcg cccgacacgg ctgggccagc 1080 ctgttcctcgacaaccacga ccagccgcgt gtcgtctccc gttggggcga cgacaccagc 1140 aagaccggccgcatccgctc cgccaaggcg ctcgcgctgc tgctgcacat gcaccgcggc 1200 actccgtatgtctaccaggg cgaggagctc ggcatgacca atgcgcactt cacctcgctc 1260 gaccagtaccgcgacctcga atccatcaac gcctaccatc aacgcgtcga ggaaaccggg 1320 atacggacatcggagaccat gatgcgatcc ctcgcccgat acggcaggga caacgcgcgc 1380 accccgatgcaatgggacga ctccacctac gccggcttca ccatgcccga cgccccggtc 1440 gaaccctggatcgccgtcaa cccgaaccac acggagatca acgccgccga cgagatcgac 1500 gaccccgactccgtgtactc gttccacaaa cggctcatcg ccctgcgtca caccgacccc 1560 gtggtcgccgccggcgacta ccgacgcgtg gaaaccggaa acgaccggat catcgccttc 1620 accagaaccctcgacgagcg aaccatcctc accgtcatca acctctcgcc cacacaggcc 1680 gcaccggccggagaactgga aacgatgccc gacggcacga tcctcatcgc caacacggac 1740 gatcccgtaggaaacctgaa aaccacggga acactcggac catgggaggc gttcgccatg 1800 gaaaccgatccggaataa 1818 6 605 PRT Bifidobacterium breve ATCC#15700 6 Met Met ThrSer Phe Asn Arg Glu Pro Leu Pro Asp Ala Val Arg Thr 1 5 10 15 Asn GlyAla Ser Pro Asn Pro Trp Trp Ser Asn Ala Val Val Tyr Gln 20 25 30 Ile TyrPro Arg Ser Phe Gln Asp Thr Asn Gly Asp Gly Phe Gly Asp 35 40 45 Leu LysGly Ile Thr Ser Arg Leu Asp Tyr Leu Ala Asp Leu Gly Val 50 55 60 Asp ValLeu Trp Leu Ser Pro Val Tyr Arg Ser Pro Gln Asp Asp Asn 65 70 75 80 GlyTyr Asp Ile Ser Asp Tyr Arg Asp Ile Asp Pro Leu Phe Gly Thr 85 90 95 LeuAsp Asp Met Asp Glu Leu Leu Ala Glu Ala His Lys Arg Gly Leu 100 105 110Lys Ile Val Met Asp Leu Val Val Asn His Thr Ser Asp Glu His Ala 115 120125 Trp Phe Glu Ala Ser Lys Asp Lys Asp Asp Pro His Ala Asp Trp Tyr 130135 140 Trp Trp Arg Pro Ala Arg Pro Gly His Glu Pro Gly Thr Pro Gly Ala145 150 155 160 Glu Pro Asn Gln Trp Gly Ser Tyr Phe Gly Gly Ser Ala TrpGlu Tyr 165 170 175 Cys Pro Glu Arg Gly Glu Tyr Tyr Leu His Gln Phe SerLys Lys Gln 180 185 190 Pro Asp Leu Asn Trp Glu Asn Pro Ala Val Arg ArgAla Val Tyr Asp 195 200 205 Met Met Asn Trp Trp Leu Asp Arg Gly Ile AspGly Phe Arg Met Asp 210 215 220 Val Ile Thr Leu Ile Ser Lys Arg Thr AspAla Asn Gly Arg Leu Pro 225 230 235 240 Gly Glu Thr Gly Ser Glu Leu GlnAsp Leu Pro Val Gly Glu Glu Gly 245 250 255 Tyr Ser Asn Pro Asn Pro PheCys Ala Asp Gly Pro Arg Gln Asp Glu 260 265 270 Phe Leu Ala Glu Met ArgArg Glu Val Phe Asp Gly Arg Asp Gly Phe 275 280 285 Leu Thr Val Gly GluAla Pro Gly Ile Thr Ala Glu Arg Asn Glu His 290 295 300 Ile Thr Asp ProAla Asn Gly Glu Leu Asp Met Leu Phe Leu Phe Glu 305 310 315 320 His MetGly Val Asp Gln Thr Pro Glu Ser Lys Trp Asp Asp Lys Pro 325 330 335 TrpThr Pro Ala Asp Leu Glu Thr Lys Leu Ala Glu Gln Gln Asp Ala 340 345 350Ile Ala Arg His Gly Trp Ala Ser Leu Phe Leu Asp Asn His Asp Gln 355 360365 Pro Arg Val Val Ser Arg Trp Gly Asp Asp Thr Ser Lys Thr Gly Arg 370375 380 Ile Arg Ser Ala Lys Ala Leu Ala Leu Leu Leu His Met His Arg Gly385 390 395 400 Thr Pro Tyr Val Tyr Gln Gly Glu Glu Leu Gly Met Thr AsnAla His 405 410 415 Phe Thr Ser Leu Asp Gln Tyr Arg Asp Leu Glu Ser IleAsn Ala Tyr 420 425 430 His Gln Arg Val Glu Glu Thr Gly Ile Arg Thr SerGlu Thr Met Met 435 440 445 Arg Ser Leu Ala Arg Tyr Gly Arg Asp Asn AlaArg Thr Pro Met Gln 450 455 460 Trp Asp Asp Ser Thr Tyr Ala Gly Phe ThrMet Pro Asp Ala Pro Val 465 470 475 480 Glu Pro Trp Ile Ala Val Asn ProAsn His Thr Glu Ile Asn Ala Ala 485 490 495 Asp Glu Ile Asp Asp Pro AspSer Val Tyr Ser Phe His Lys Arg Leu 500 505 510 Ile Ala Leu Arg His ThrAsp Pro Val Val Ala Ala Gly Asp Tyr Arg 515 520 525 Arg Val Glu Thr GlyAsn Asp Arg Ile Ile Ala Phe Thr Arg Thr Leu 530 535 540 Asp Glu Arg ThrIle Leu Thr Val Ile Asn Leu Ser Pro Thr Gln Ala 545 550 555 560 Ala ProAla Gly Glu Leu Glu Thr Met Pro Asp Gly Thr Ile Leu Ile 565 570 575 AlaAsn Thr Asp Asp Pro Val Gly Asn Leu Lys Thr Thr Gly Thr Leu 580 585 590Gly Pro Trp Glu Ala Phe Ala Met Glu Thr Asp Pro Glu 595 600 605 7 39 DNAArtificial Sequence Primer 7 gcatgcggat ccatgcaaaa acattggtgg cacaaggca39 8 44 DNA Artificial Sequence Primer 8 ggtaccgtcg acttagtttatcttaataca aaaagcatcc caag 44 9 39 DNA Artificial Sequence Primer 9gacgtatatg atatccgcgc tagcagagga tgtgctgcc 39 10 17 DNA ArtificialSequence Primer 10 gaattcgagc tcggtac 17 11 39 DNA Artificial SequencePrimer 11 gacgtatatg atatccgcgc tagcacccgg cagactgat 39 12 29 DNAArtificial Sequence Primer 12 atgcatggta ccgatctaac attttcccc 29 13 36DNA Artificial Sequence Primer 13 gagtctgcta gcgcgatgca aaaacattggtggcac 36 14 42 DNA Artificial Sequence Primer 14 cgcggatccg ctagcgcgatgatgacctct ttcaaccgtg aa 42 15 36 DNA Artificial Sequence Primer 15gagtctaagc ttttattccg gatcggtttc catggc 36 16 1611 DNA Streptococcusmutans ATCC#25175D 16 atgcaaaaac attggtggca caaggcaact gtttatcaaatttatccaaa atcttttatg 60 gatacaaatg gtgatggaat tggtgatctc aaaggtattacgagtaaatt ggattatttg 120 caaaagttag gggttatggc tatttggcta tctccagtttatgatagccc catggatgac 180 aatggctatg acattgcgaa ctatgaagca attgcggatatttttggcaa tatggctgat 240 atggataatt tgctgacgca ggcaaaaatg cgcgacataaaaatcattat ggatctagtg 300 gttaatcata cctcagatga acatacttgg tttattgaagcacgtgagca tccagacagt 360 tctgaacgcg attattatat ttggtgtgac cagccaaatgatttggaatc tattttcggt 420 ggttctgctt ggcagtatga tgataagtcc gatcaatattatttgcattt ttttagtaag 480 aagcagccag atctaaactg ggaaaacgca aacttacgtcagaagattta tgatatgatg 540 aatttctgga ttgataaagg tattggcggc tttcggatggacgtcattga tatgattggg 600 aaaattcctg ctcagcatat tgtcagtaac ggaccaaaattgcatgctta tcttaaggag 660 atgaatgccg ctagttttgg tcaacatgat ctgctgactgtgggggaaac ttggggagca 720 acgcctgaga ttgcgaagca atattcaaat ccagtcaatcacgaactctc tatgattttt 780 caatttgaac atattggtct tcagcataaa ccagaagctcctaaatggga ttatgtgaag 840 gaacttaatg ttcctgcttt aaaaacaatc tttaataaatggcagactga gttggaatta 900 ggacaggggt ggaattcgtt attctggaat aaccatgacctgcctcgtgt tttatcaatc 960 tggggaaata cgggcaaata tcgtgagaag tctgctaaagcactggctat tcttcttcac 1020 cttatgcgtg ggacacctta tatttatcaa ggtgaagagattgggatgac caattatcct 1080 tttaaagatt taaatgaact tgatgatatt gaatcacttaattatgctaa ggaagctttt 1140 acaaatggta agtctatgga aactatcatg gacagtattcgtatgattgg ccgtgataat 1200 gccagaacac ctatgcaatg ggatgcttct caaaatgccggattttcaac agcggataaa 1260 acatggctgc cagttaatcc aaactataaa gacatcaatgttcaagcagc tctgaaaaat 1320 tccaattcta tcttttacac ctatcaacaa ctcattcagcttcgaaaaga aaatgattgg 1380 ctagtagatg ccgattttga attgctccct acagcggacaaagtatttgc ctatttacga 1440 aaggtaagag aagaaaggta tcttatagtg gtcaatgtttcagatcagga agaagttcta 1500 gagattgatg ttgacaaaca agaaactctc attagcaatacaaatgaaag cgctgctctt 1560 gccaatcaca aactccagcc ttgggatgct ttttgtattaagataaacta a 1611 17 536 PRT Streptococcus mutans ATCC#25175D 17 Met GlnLys His Trp Trp His Lys Ala Thr Val Tyr Gln Ile Tyr Pro 1 5 10 15 LysSer Phe Met Asp Thr Asn Gly Asp Gly Ile Gly Asp Leu Lys Gly 20 25 30 IleThr Ser Lys Leu Asp Tyr Leu Gln Lys Leu Gly Val Met Ala Ile 35 40 45 TrpLeu Ser Pro Val Tyr Asp Ser Pro Met Asp Asp Asn Gly Tyr Asp 50 55 60 IleAla Asn Tyr Glu Ala Ile Ala Asp Ile Phe Gly Asn Met Ala Asp 65 70 75 80Met Asp Asn Leu Leu Thr Gln Ala Lys Met Arg Asp Ile Lys Ile Ile 85 90 95Met Asp Leu Val Val Asn His Thr Ser Asp Glu His Thr Trp Phe Ile 100 105110 Glu Ala Arg Glu His Pro Asp Ser Ser Glu Arg Asp Tyr Tyr Ile Trp 115120 125 Cys Asp Gln Pro Asn Asp Leu Glu Ser Ile Phe Gly Gly Ser Ala Trp130 135 140 Gln Tyr Asp Asp Lys Ser Asp Gln Tyr Tyr Leu His Phe Phe SerLys 145 150 155 160 Lys Gln Pro Asp Leu Asn Trp Glu Asn Ala Asn Leu ArgGln Lys Ile 165 170 175 Tyr Asp Met Met Asn Phe Trp Ile Asp Lys Gly IleGly Gly Phe Arg 180 185 190 Met Asp Val Ile Asp Met Ile Gly Lys Ile ProAla Gln His Ile Val 195 200 205 Ser Asn Gly Pro Lys Leu His Ala Tyr LeuLys Glu Met Asn Ala Ala 210 215 220 Ser Phe Gly Gln His Asp Leu Leu ThrVal Gly Glu Thr Trp Gly Ala 225 230 235 240 Thr Pro Glu Ile Ala Lys GlnTyr Ser Asn Pro Val Asn His Glu Leu 245 250 255 Ser Met Ile Phe Gln PheGlu His Ile Gly Leu Gln His Lys Pro Glu 260 265 270 Ala Pro Lys Trp AspTyr Val Lys Glu Leu Asn Val Pro Ala Leu Lys 275 280 285 Thr Ile Phe AsnLys Trp Gln Thr Glu Leu Glu Leu Gly Gln Gly Trp 290 295 300 Asn Ser LeuPhe Trp Asn Asn His Asp Leu Pro Arg Val Leu Ser Ile 305 310 315 320 TrpGly Asn Thr Gly Lys Tyr Arg Glu Lys Ser Ala Lys Ala Leu Ala 325 330 335Ile Leu Leu His Leu Met Arg Gly Thr Pro Tyr Ile Tyr Gln Gly Glu 340 345350 Glu Ile Gly Met Thr Asn Tyr Pro Phe Lys Asp Leu Asn Glu Leu Asp 355360 365 Asp Ile Glu Ser Leu Asn Tyr Ala Lys Glu Ala Phe Thr Asn Gly Lys370 375 380 Ser Met Glu Thr Ile Met Asp Ser Ile Arg Met Ile Gly Arg AspAsn 385 390 395 400 Ala Arg Thr Pro Met Gln Trp Asp Ala Ser Gln Asn AlaGly Phe Ser 405 410 415 Thr Ala Asp Lys Thr Trp Leu Pro Val Asn Pro AsnTyr Lys Asp Ile 420 425 430 Asn Val Gln Ala Ala Leu Lys Asn Ser Asn SerIle Phe Tyr Thr Tyr 435 440 445 Gln Gln Leu Ile Gln Leu Arg Lys Glu AsnAsp Trp Leu Val Asp Ala 450 455 460 Asp Phe Glu Leu Leu Pro Thr Ala AspLys Val Phe Ala Tyr Leu Arg 465 470 475 480 Lys Val Arg Glu Glu Arg TyrLeu Ile Val Val Asn Val Ser Asp Gln 485 490 495 Glu Glu Val Leu Glu IleAsp Val Asp Lys Gln Glu Thr Leu Ile Ser 500 505 510 Asn Thr Asn Glu SerAla Ala Leu Ala Asn His Lys Leu Gln Pro Trp 515 520 525 Asp Ala Phe CysIle Lys Ile Asn 530 535 18 35 DNA Artificial Sequence Primer 18tccgagctca tgggtttagg taagaaattg tctgt 35 19 37 DNA Artificial SequencePrimer 19 accttaatta aggttattcc ggatcggttt ccatggc 37 20 89 DNAArtificial Sequence Primer 20 gtgatagctg tcgtaaagct gttaccgactggcgaagatt tcgccagtca cgtctaccct 60 tgttataccg tgtaggctgg agctgcttc 8921 93 DNA Artificial Sequence Primer 21 tcagaacagc cccaacggtt tatccgagtagctcaccagc aggcacttgg tttgctggta 60 atgctccagc ttattccgga tcggtttcca tgg93 22 89 DNA Artificial Sequence Primer 22 atgtcagtac ccgttcaacatcctatgtat atcgatggac agtttgttac ctggcgtgga 60 gacgcatggg tgtaggctggagctgcttc 89 23 93 DNA Artificial Sequence Primer 23 ttaagactgtaaataaacca cctgggtctg cagatattca tgcaagccat gtttaccatc 60 tgcgccgccattattccgga tcggtttcca tgg 93 24 19 PRT Artificial Sequence NPR Signal 24Met Asn Lys Glu Pro Thr Met Thr Thr Phe Asn Arg Thr Ile Pro Asp 1 5 1015 Ala Ile Arg 25 14 PRT Artificial Sequence Bifidobacterium breveSignal 25 Met Ser Leu Thr Ile Ser Leu Pro Gly Val Gln Ala Ser Ala 1 5 1026 62 DNA Artificial Sequence Bifidobacterium breve signal 26 atgaataaggagccaacgat gactactttc aaccgcacaa taattcccga cgccattcgc 60 ac 62 27 42DNA Artificial Sequence NPR Signal 27 atgagtttaa ccatcagtct gccgggtgttcaggctagcg cg 42 28 1877 DNA Bifidobacterium breve 28 atgaataaggagccaacgat gactactttc aaccgcacaa taattcccga cgccattcgc 60 acatgaccgccaacaacctc aatgacgact ggtggaagca ggccgtcgtt taccagattt 120 acccgcgcagcttcaaggac gttaacggcg acggcatcgg cgacatcgcc ggcgttaccg 180 agaaaatggactacctgaaa aacctcggcg tggacgccat ctggctctcc ccgttctacc 240 cctccgatctggcggacggc ggctacgacg tgatcgacta ccgcaacgtc gacccgcgac 300 tgggcaccatggacgacttc gacgccatgg ccaaagccgc gcatgaggcc ggcatcaagg 360 tgatcgtggacatcgtgccc aatcacaccg ccgacaagca cgtgttcttc aaggaagccc 420 tcgccgccgagcccggctcc ccggcgcgcg accgctacat cttccgcgac ggccgcggcg 480 agcacggcgaactgccgccc aacgactggc agtccttctt cggcggcccg gcctgggctc 540 gcgtggccgacggccagtgg tatctgcacc tgttcgacaa ggcgcaaccg gacgtcaact 600 ggaagaacccggacatccac gaggaattca agaaaaccct gcgcttctgg tccgaccacg 660 gcaccgacggcttccgcatc gacgtggcgc acggtctggc caaagacctt gaatccaagc 720 cgctggaggagctcggccgc gaatacagcg tggtcggcgt gctgaatcac gacttcagcc 780 atccgctgttcgaccgccgc gaagtgcacg acatctaccg cgaatggcgc aaggtgttca 840 acgagtacgacccgccgcgc tttgccgtgg ccgaggcgtg ggtggtaccc gagcaccagc 900 acctgtatgcctcgatggat gagctggggc agtccttcaa cttcgacttt gcgcaggcca 960 gctggtatgccgatgagttc cgcgcagcca tcgccgcggg tctcaaggcc gctgccgaaa 1020 ccggcggttccaccaccacg tgggtgatga acaaccatga cgtgccgcgc agcccctccc 1080 gctatggtctaccgcaggtc aagggcgcgc cttaccacca gctgccgcac gactggctgc 1140 tgcgcaacggcaccacgtat cccgaggatc gcgagcttgg cacccgccgc gcccgcgccg 1200 ccgctttgatggagctcggc ctgcccggcg ccgcctatat ctatcagggc gaggagctgg 1260 gcctgtttgaagtggccgat attccgtggg atcgactgga agatccgacc gctttccaca 1320 ccgctcaggccacgatggac aagggccgag acggctgccg cgtgccgatt ccgtggaccg 1380 ctgcaaacgaaccgaccttg gctgatttca gccgcccgat cccggccgat gacggcaccg 1440 gcgagaaccacgtgccgctg tgcgccgccg gccagttcgg cacgggcgct tccttcggct 1500 tctcgccggctacgcgcgct gagggcgtga cgccggccgc cgacccgcac ctgccgcagc 1560 cgttgtggttcaaggattac gcggtggacg tggagcaggc cgacccggat tcaatgctcg 1620 cgctgtatcatgcggcgttg gcgattcgtc aggagtcgct gaccgccacg cgtgacacca 1680 ccgctgagcaggtggatatg gggccggacg tggtggccta cacccgcgcg gcggttggtg 1740 gccgcacgttcacctcgatc accaacttcg gcaccgagcc ggtggagctg cctggaggct 1800 ccgtcgtgctgacgtccggc ccgctgaccc ccgacggcca gctccccacc gacacttctg 1860 cgtgggttatcaagtag 1877 29 624 PRT Bifidobacterium breve 29 Met Asn Lys Glu Pro ThrMet Thr Thr Phe Asn Arg Thr Ile Ile Pro 1 5 10 15 Asp Ala Ile Arg MetThr Ala Asn Asn Leu Asn Asp Asp Trp Trp Lys 20 25 30 Gln Ala Val Val TyrGln Ile Tyr Pro Arg Ser Phe Lys Asp Val Asn 35 40 45 Gly Asp Gly Ile GlyAsp Ile Ala Gly Val Thr Glu Lys Met Asp Tyr 50 55 60 Leu Lys Asn Leu GlyVal Asp Ala Ile Trp Leu Ser Pro Phe Tyr Pro 65 70 75 80 Ser Asp Leu AlaAsp Gly Gly Tyr Asp Val Ile Asp Tyr Arg Asn Val 85 90 95 Asp Pro Arg LeuGly Thr Met Asp Asp Phe Asp Ala Met Ala Lys Ala 100 105 110 Ala His GluAla Gly Ile Lys Val Ile Val Asp Ile Val Pro Asn His 115 120 125 Thr AlaAsp Lys His Val Phe Phe Lys Glu Ala Leu Ala Ala Glu Pro 130 135 140 GlySer Pro Ala Arg Asp Arg Tyr Ile Phe Arg Asp Gly Arg Gly Glu 145 150 155160 His Gly Glu Leu Pro Pro Asn Asp Trp Gln Ser Phe Phe Gly Gly Pro 165170 175 Ala Trp Ala Arg Val Ala Asp Gly Gln Trp Tyr Leu His Leu Phe Asp180 185 190 Lys Ala Gln Pro Asp Val Asn Trp Lys Asn Pro Asp Ile His GluGlu 195 200 205 Phe Lys Lys Thr Leu Arg Phe Trp Ser Asp His Gly Thr AspGly Phe 210 215 220 Arg Ile Asp Val Ala His Gly Leu Ala Lys Asp Leu GluSer Lys Pro 225 230 235 240 Leu Glu Glu Leu Gly Arg Glu Tyr Ser Val ValGly Val Leu Asn His 245 250 255 Asp Phe Ser His Pro Leu Phe Asp Arg ArgGlu Val His Asp Ile Tyr 260 265 270 Arg Glu Trp Arg Lys Val Phe Asn GluTyr Asp Pro Pro Arg Phe Ala 275 280 285 Val Ala Glu Ala Trp Val Val ProGlu His Gln His Leu Tyr Ala Ser 290 295 300 Met Asp Glu Leu Gly Gln SerPhe Asn Phe Asp Phe Ala Gln Ala Ser 305 310 315 320 Trp Tyr Ala Asp GluPhe Arg Ala Ala Ile Ala Ala Gly Leu Lys Ala 325 330 335 Ala Ala Glu ThrGly Gly Ser Thr Thr Thr Trp Val Met Asn Asn His 340 345 350 Asp Val ProArg Ser Pro Ser Arg Tyr Gly Leu Pro Gln Val Lys Gly 355 360 365 Ala ProTyr His Gln Leu Pro His Asp Trp Leu Leu Arg Asn Gly Thr 370 375 380 ThrTyr Pro Glu Asp Arg Glu Leu Gly Thr Arg Arg Ala Arg Ala Ala 385 390 395400 Ala Leu Met Glu Leu Gly Leu Pro Gly Ala Ala Tyr Ile Tyr Gln Gly 405410 415 Glu Glu Leu Gly Leu Phe Glu Val Ala Asp Ile Pro Trp Asp Arg Leu420 425 430 Glu Asp Pro Thr Ala Phe His Thr Ala Gln Ala Thr Met Asp LysGly 435 440 445 Arg Asp Gly Cys Arg Val Pro Ile Pro Trp Thr Ala Ala AsnGlu Pro 450 455 460 Thr Leu Ala Asp Phe Ser Arg Pro Ile Pro Ala Asp AspGly Thr Gly 465 470 475 480 Glu Asn His Val Pro Leu Cys Ala Ala Gly GlnPhe Gly Thr Gly Ala 485 490 495 Ser Phe Gly Phe Ser Pro Ala Thr Arg AlaGlu Gly Val Thr Pro Ala 500 505 510 Ala Asp Pro His Leu Pro Gln Pro LeuTrp Phe Lys Asp Tyr Ala Val 515 520 525 Asp Val Glu Gln Ala Asp Pro AspSer Met Leu Ala Leu Tyr His Ala 530 535 540 Ala Leu Ala Ile Arg Gln GluSer Leu Thr Ala Thr Arg Asp Thr Thr 545 550 555 560 Ala Glu Gln Val AspMet Gly Pro Asp Val Val Ala Tyr Thr Arg Ala 565 570 575 Ala Val Gly GlyArg Thr Phe Thr Ser Ile Thr Asn Phe Gly Thr Glu 580 585 590 Pro Val GluLeu Pro Gly Gly Ser Val Val Leu Thr Ser Gly Pro Leu 595 600 605 Thr ProAsp Gly Gln Leu Pro Thr Asp Thr Ser Ala Trp Val Ile Lys 610 615 620 301611 DNA Bifidobacterium breve 30 atgcaaaaac attggtggca caaggcaactgtttatcaaa tttatccaaa atcttttatg 60 gatacaaatg gtgatggaat tggtgatctcaaaggtatta cgagtaaatt ggattatttg 120 caaaagttag gggttatggc tatttggctatctccagttt atgatagccc catggatgac 180 aatggctatg acattgcgaa ctatgaagcaattgcggata tttttggcaa tatggctgat 240 atggataatt tgctgacgca ggcaaaaatgcgcgacataa aaatcattat ggatctagtg 300 gttaatcata cctcagatga acatacttggtttattgaag cacgtgagca tccagacagt 360 tctgaacgcg attattatat ttggtgtgaccagccaaatg atttggaatc tattttcggt 420 ggttctgctt ggcagtatga tgataagtccgatcaatatt atttgcattt ttttagtaag 480 aagcagccag atctaaactg ggaaaacgcaaacttacgtc agaagattta tgatatgatg 540 aatttctgga ttgataaagg tattggcggctttcggatgg acgtcattga tatgattggg 600 aaaattcctg ctcagcatat tgtcagtaacggaccaaaat tgcatgctta tcttaaggag 660 atgaatgccg ctagttttgg tcaacatgatctgctgactg tgggggaaac ttggggagca 720 acgcctgaga ttgcgaagca atattcaaatccagtcaatc acgaactctc tatgattttt 780 caatttgaac atattggtct tcagcataaaccagaagctc ctaaatggga ttatgtgaag 840 gaacttaatg ttcctgcttt aaaaacaatctttaataaat ggcagactga gttggaatta 900 ggacaggggt ggaattcgtt attctggaataaccatgacc tgcctcgtgt tttatcaatc 960 tggggaaata cgggcaaata tcgtgagaagtctgctaaag cactggctat tcttcttcac 1020 cttatgcgtg ggacacctta tatttatcaaggtgaagaga ttgggatgac caattatcct 1080 tttaaagatt taaatgaact tgatgatattgaatcactta attatgctaa ggaagctttt 1140 acaaatggta agtctatgga aactatcatggacagtattc gtatgattgg ccgtgataat 1200 gccagaacac ctatgcaatg ggatgcttctcaaaatgccg gattttcaac agcggataaa 1260 acatggctgc cagttaatcc aaactataaagacatcaatg ttcaagcagc tctgaaaaat 1320 tccaattcta tcttttacac ctatcaacaactcattcagc ttcgaaaaga aaatgattgg 1380 ctagtagatg ccgattttga attgctccctacagcggaca aagtatttgc ctatttacga 1440 aaggtaagag aagaaaggta tcttatagtggtcaatgttt cagatcagga agaagttcta 1500 gagattgatg ttgacaaaca agaaactctcattagcaata caaatgaaag cgctgctctt 1560 gccaatcaca aactccagcc ttgggatgctttttgtatta agataaacta a 1611 31 536 PRT Bifidobacterium breve 31 Met GlnLys His Trp Trp His Lys Ala Thr Val Tyr Gln Ile Tyr Pro 1 5 10 15 LysSer Phe Met Asp Thr Asn Gly Asp Gly Ile Gly Asp Leu Lys Gly 20 25 30 IleThr Ser Lys Leu Asp Tyr Leu Gln Lys Leu Gly Val Met Ala Ile 35 40 45 TrpLeu Ser Pro Val Tyr Asp Ser Pro Met Asp Asp Asn Gly Tyr Asp 50 55 60 IleAla Asn Tyr Glu Ala Ile Ala Asp Ile Phe Gly Asn Met Ala Asp 65 70 75 80Met Asp Asn Leu Leu Thr Gln Ala Lys Met Arg Asp Ile Lys Ile Ile 85 90 95Met Asp Leu Val Val Asn His Thr Ser Asp Glu His Thr Trp Phe Ile 100 105110 Glu Ala Arg Glu His Pro Asp Ser Ser Glu Arg Asp Tyr Tyr Ile Trp 115120 125 Cys Asp Gln Pro Asn Asp Leu Glu Ser Ile Phe Gly Gly Ser Ala Trp130 135 140 Gln Tyr Asp Asp Lys Ser Asp Gln Tyr Tyr Leu His Phe Phe SerLys 145 150 155 160 Lys Gln Pro Asp Leu Asn Trp Glu Asn Ala Asn Leu ArgGln Lys Ile 165 170 175 Tyr Asp Met Met Asn Phe Trp Ile Asp Lys Gly IleGly Gly Phe Arg 180 185 190 Met Asp Val Ile Asp Met Ile Gly Lys Ile ProAla Gln His Ile Val 195 200 205 Ser Asn Gly Pro Lys Leu His Ala Tyr LeuLys Glu Met Asn Ala Ala 210 215 220 Ser Phe Gly Gln His Asp Leu Leu ThrVal Gly Glu Thr Trp Gly Ala 225 230 235 240 Thr Pro Glu Ile Ala Lys GlnTyr Ser Asn Pro Val Asn His Glu Leu 245 250 255 Ser Met Ile Phe Gln PheGlu His Ile Gly Leu Gln His Lys Pro Glu 260 265 270 Ala Pro Lys Trp AspTyr Val Lys Glu Leu Asn Val Pro Ala Leu Lys 275 280 285 Thr Ile Phe AsnLys Trp Gln Thr Glu Leu Glu Leu Gly Gln Gly Trp 290 295 300 Asn Ser LeuPhe Trp Asn Asn His Asp Leu Pro Arg Val Leu Ser Ile 305 310 315 320 TrpGly Asn Thr Gly Lys Tyr Arg Glu Lys Ser Ala Lys Ala Leu Ala 325 330 335Ile Leu Leu His Leu Met Arg Gly Thr Pro Tyr Ile Tyr Gln Gly Glu 340 345350 Glu Ile Gly Met Thr Asn Tyr Pro Phe Lys Asp Leu Asn Glu Leu Asp 355360 365 Asp Ile Glu Ser Leu Asn Tyr Ala Lys Glu Ala Phe Thr Asn Gly Lys370 375 380 Ser Met Glu Thr Ile Met Asp Ser Ile Arg Met Ile Gly Arg AspAsn 385 390 395 400 Ala Arg Thr Pro Met Gln Trp Asp Ala Ser Gln Asn AlaGly Phe Ser 405 410 415 Thr Ala Asp Lys Thr Trp Leu Pro Val Asn Pro AsnTyr Lys Asp Ile 420 425 430 Asn Val Gln Ala Ala Leu Lys Asn Ser Asn SerIle Phe Tyr Thr Tyr 435 440 445 Gln Gln Leu Ile Gln Leu Arg Lys Glu AsnAsp Trp Leu Val Asp Ala 450 455 460 Asp Phe Glu Leu Leu Pro Thr Ala AspLys Val Phe Ala Tyr Leu Arg 465 470 475 480 Lys Val Arg Glu Glu Arg TyrLeu Ile Val Val Asn Val Ser Asp Gln 485 490 495 Glu Glu Val Leu Glu IleAsp Val Asp Lys Gln Glu Thr Leu Ile Ser 500 505 510 Asn Thr Asn Glu SerAla Ala Leu Ala Asn His Lys Leu Gln Pro Trp 515 520 525 Asp Ala Phe CysIle Lys Ile Asn 530 535 32 1880 DNA Bifidobacterium breve 32 atgaataaggagccaacgat gactactttc aaccgcacaa taattcccga cgccattcgc 60 acatgatgacctctttcaac cgtgaacccc tgcccgacgc cgtccgcacg aatggcgcct 120 ccccgaacccgtggtggtcg aacgccgtcg tctaccagat ttacccacgt tccttccagg 180 acacgaacggcgatggtttc ggagatctta agggcattac ttcccgcctc gactatcttg 240 ccgacctcggcgtggatgtg ctgtggctct ccccggtcta caggtccccg caagacgaca 300 acggctacgacatctccgac taccgggaca tcgacccgct gttcggcacg ctcgacgaca 360 tggacgagctgctcgccgaa gcgcacaagc gcggcctcaa gatcgtgatg gacctggtcg 420 tcaaccacacctccgacgag cacgcgtggt tcgaggcgtc gaaggacaag gacgacccgc 480 acgccgactggtactggtgg cgtcccgccc gccccggcca cgagcccggc acgcccggcg 540 ccgagccgaaccagtggggc tcctacttcg gcggctccgc atgggaatat tgccccgagc 600 gtggtgagtactatctccac cagttctcga agaagcagcc ggacctcaac tgggagaacc 660 cggccgtacgccgagccgtg tacgacatga tgaactggtg gctcgatcgc ggcatcgacg 720 gcttccgcatggacgtcatc accctgatct ccaagcgcac ggatgcaaac ggcaggctgc 780 ccggcgagaccggttccgag ctgcaggacc tgccggtggg ggaggagggc tactccaacc 840 cgaacccgttctgcgccgac ggtccgcgtc aggacgagtt cctcgccgag atgcgccgcg 900 aggtgttcgacgggcgtgac ggcttcctca ccgtcggcga ggcccccggc atcaccgccg 960 aacgcaacgagcacatcacc gaccccgcca acggcgagct ggatatgctc ttcctgttcg 1020 aacacatgggcgtcgaccaa acccccgaat cgaaatggga cgacaaacca tggacgccgg 1080 ccgacctcgaaaccaagctc gccgaacaac aggacgccat cgcccgacac ggctgggcca 1140 gcctgttcctcgacaaccac gaccagccgc gtgtcgtctc ccgttggggc gacgacacca 1200 gcaagaccggccgcatccgc tccgccaagg cgctcgcgct gctgctgcac atgcaccgcg 1260 gcactccgtatgtctaccag ggcgaggagc tcggcatgac caatgcgcac ttcacctcgc 1320 tcgaccagtaccgcgacctc gaatccatca acgcctacca tcaacgcgtc gaggaaaccg 1380 ggatacggacatcggagacc atgatgcgat ccctcgcccg atacggcagg gacaacgcgc 1440 gcaccccgatgcaatgggac gactccacct acgccggctt caccatgccc gacgccccgg 1500 tcgaaccctggatcgccgtc aacccgaacc acacggagat caacgccgcc gacgagatcg 1560 acgaccccgactccgtgtac tcgttccaca aacggctcat cgccctgcgt cacaccgacc 1620 ccgtggtcgccgccggcgac taccgacgcg tggaaaccgg aaacgaccgg atcatcgcct 1680 tcaccagaaccctcgacgag cgaaccatcc tcaccgtcat caacctctcg cccacacagg 1740 ccgcaccggccggagaactg gaaacgatgc ccgacggcac gatcctcatc gccaacacgg 1800 acgatcccgtaggaaacctg aaaaccacgg gaacactcgg accatgggag gcgttcgcca 1860 tggaaaccgatccggaataa 1880 33 625 PRT Bifidobacterium breve 33 Met Asn Lys Glu ProThr Met Thr Thr Phe Asn Arg Thr Ile Ile Pro 1 5 10 15 Asp Ala Ile ArgMet Met Thr Ser Phe Asn Arg Glu Pro Leu Pro Asp 20 25 30 Ala Val Arg ThrAsn Gly Ala Ser Pro Asn Pro Trp Trp Ser Asn Ala 35 40 45 Val Val Tyr GlnIle Tyr Pro Arg Ser Phe Gln Asp Thr Asn Gly Asp 50 55 60 Gly Phe Gly AspLeu Lys Gly Ile Thr Ser Arg Leu Asp Tyr Leu Ala 65 70 75 80 Asp Leu GlyVal Asp Val Leu Trp Leu Ser Pro Val Tyr Arg Ser Pro 85 90 95 Gln Asp AspAsn Gly Tyr Asp Ile Ser Asp Tyr Arg Asp Ile Asp Pro 100 105 110 Leu PheGly Thr Leu Asp Asp Met Asp Glu Leu Leu Ala Glu Ala His 115 120 125 LysArg Gly Leu Lys Ile Val Met Asp Leu Val Val Asn His Thr Ser 130 135 140Asp Glu His Ala Trp Phe Glu Ala Ser Lys Asp Lys Asp Asp Pro His 145 150155 160 Ala Asp Trp Tyr Trp Trp Arg Pro Ala Arg Pro Gly His Glu Pro Gly165 170 175 Thr Pro Gly Ala Glu Pro Asn Gln Trp Gly Ser Tyr Phe Gly GlySer 180 185 190 Ala Trp Glu Tyr Cys Pro Glu Arg Gly Glu Tyr Tyr Leu HisGln Phe 195 200 205 Ser Lys Lys Gln Pro Asp Leu Asn Trp Glu Asn Pro AlaVal Arg Arg 210 215 220 Ala Val Tyr Asp Met Met Asn Trp Trp Leu Asp ArgGly Ile Asp Gly 225 230 235 240 Phe Arg Met Asp Val Ile Thr Leu Ile SerLys Arg Thr Asp Ala Asn 245 250 255 Gly Arg Leu Pro Gly Glu Thr Gly SerGlu Leu Gln Asp Leu Pro Val 260 265 270 Gly Glu Glu Gly Tyr Ser Asn ProAsn Pro Phe Cys Ala Asp Gly Pro 275 280 285 Arg Gln Asp Glu Phe Leu AlaGlu Met Arg Arg Glu Val Phe Asp Gly 290 295 300 Arg Asp Gly Phe Leu ThrVal Gly Glu Ala Pro Gly Ile Thr Ala Glu 305 310 315 320 Arg Asn Glu HisIle Thr Asp Pro Ala Asn Gly Glu Leu Asp Met Leu 325 330 335 Phe Leu PheGlu His Met Gly Val Asp Gln Thr Pro Glu Ser Lys Trp 340 345 350 Asp AspLys Pro Trp Thr Pro Ala Asp Leu Glu Thr Lys Leu Ala Glu 355 360 365 GlnGln Asp Ala Ile Ala Arg His Gly Trp Ala Ser Leu Phe Leu Asp 370 375 380Asn His Asp Gln Pro Arg Val Val Ser Arg Trp Gly Asp Asp Thr Ser 385 390395 400 Lys Thr Gly Arg Ile Arg Ser Ala Lys Ala Leu Ala Leu Leu Leu His405 410 415 Met His Arg Gly Thr Pro Tyr Val Tyr Gln Gly Glu Glu Leu GlyMet 420 425 430 Thr Asn Ala His Phe Thr Ser Leu Asp Gln Tyr Arg Asp LeuGlu Ser 435 440 445 Ile Asn Ala Tyr His Gln Arg Val Glu Glu Thr Gly IleArg Thr Ser 450 455 460 Glu Thr Met Met Arg Ser Leu Ala Arg Tyr Gly ArgAsp Asn Ala Arg 465 470 475 480 Thr Pro Met Gln Trp Asp Asp Ser Thr TyrAla Gly Phe Thr Met Pro 485 490 495 Asp Ala Pro Val Glu Pro Trp Ile AlaVal Asn Pro Asn His Thr Glu 500 505 510 Ile Asn Ala Ala Asp Glu Ile AspAsp Pro Asp Ser Val Tyr Ser Phe 515 520 525 His Lys Arg Leu Ile Ala LeuArg His Thr Asp Pro Val Val Ala Ala 530 535 540 Gly Asp Tyr Arg Arg ValGlu Thr Gly Asn Asp Arg Ile Ile Ala Phe 545 550 555 560 Thr Arg Thr LeuAsp Glu Arg Thr Ile Leu Thr Val Ile Asn Leu Ser 565 570 575 Pro Thr GlnAla Ala Pro Ala Gly Glu Leu Glu Thr Met Pro Asp Gly 580 585 590 Thr IleLeu Ile Ala Asn Thr Asp Asp Pro Val Gly Asn Leu Lys Thr 595 600 605 ThrGly Thr Leu Gly Pro Trp Glu Ala Phe Ala Met Glu Thr Asp Pro 610 615 620Glu 625 34 1673 DNA Bifidobacterium breve + Streptococcus mutans 34atgaataagg agccaacgat gactactttc aaccgcacaa taattcccga cgccattcgc 60acatgcaaaa acattggtgg cacaaggcaa ctgtttatca aatttatcca aaatctttta 120tggatacaaa tggtgatgga attggtgatc tcaaaggtat tacgagtaaa ttggattatt 180tgcaaaagtt aggggttatg gctatttggc tatctccagt ttatgatagc cccatggatg 240acaatggcta tgacattgcg aactatgaag caattgcgga tatttttggc aatatggctg 300atatggataa tttgctgacg caggcaaaaa tgcgcgacat aaaaatcatt atggatctag 360tggttaatca tacctcagat gaacatactt ggtttattga agcacgtgag catccagaca 420gttctgaacg cgattattat atttggtgtg accagccaaa tgatttggaa tctattttcg 480gtggttctgc ttggcagtat gatgataagt ccgatcaata ttatttgcat ttttttagta 540agaagcagcc agatctaaac tgggaaaacg caaacttacg tcagaagatt tatgatatga 600tgaatttctg gattgataaa ggtattggcg gctttcggat ggacgtcatt gatatgattg 660ggaaaattcc tgctcagcat attgtcagta acggaccaaa attgcatgct tatcttaagg 720agatgaatgc cgctagtttt ggtcaacatg atctgctgac tgtgggggaa acttggggag 780caacgcctga gattgcgaag caatattcaa atccagtcaa tcacgaactc tctatgattt 840ttcaatttga acatattggt cttcagcata aaccagaagc tcctaaatgg gattatgtga 900aggaacttaa tgttcctgct ttaaaaacaa tctttaataa atggcagact gagttggaat 960taggacaggg gtggaattcg ttattctgga ataaccatga cctgcctcgt gttttatcaa 1020tctggggaaa tacgggcaaa tatcgtgaga agtctgctaa agcactggct attcttcttc 1080accttatgcg tgggacacct tatatttatc aaggtgaaga gattgggatg accaattatc 1140cttttaaaga tttaaatgaa cttgatgata ttgaatcact taattatgct aaggaagctt 1200ttacaaatgg taagtctatg gaaactatca tggacagtat tcgtatgatt ggccgtgata 1260atgccagaac acctatgcaa tgggatgctt ctcaaaatgc cggattttca acagcggata 1320aaacatggct gccagttaat ccaaactata aagacatcaa tgttcaagca gctctgaaaa 1380attccaattc tatcttttac acctatcaac aactcattca gcttcgaaaa gaaaatgatt 1440ggctagtaga tgccgatttt gaattgctcc ctacagcgga caaagtattt gcctatttac 1500gaaaggtaag agaagaaagg tatcttatag tggtcaatgt ttcagatcag gaagaagttc 1560tagagattga tgttgacaaa caagaaactc tcattagcaa tacaaatgaa agcgctgctc 1620ttgccaatca caaactccag ccttgggatg ctttttgtat taagataaac taa 1673 35 556PRT Bifidobacterium breve + Streptococcus mutans 35 Met Asn Lys Glu ProThr Met Thr Thr Phe Asn Arg Thr Ile Ile Pro 1 5 10 15 Asp Ala Ile ArgMet Gln Lys His Trp Trp His Lys Ala Thr Val Tyr 20 25 30 Gln Ile Tyr ProLys Ser Phe Met Asp Thr Asn Gly Asp Gly Ile Gly 35 40 45 Asp Leu Lys GlyIle Thr Ser Lys Leu Asp Tyr Leu Gln Lys Leu Gly 50 55 60 Val Met Ala IleTrp Leu Ser Pro Val Tyr Asp Ser Pro Met Asp Asp 65 70 75 80 Asn Gly TyrAsp Ile Ala Asn Tyr Glu Ala Ile Ala Asp Ile Phe Gly 85 90 95 Asn Met AlaAsp Met Asp Asn Leu Leu Thr Gln Ala Lys Met Arg Asp 100 105 110 Ile LysIle Ile Met Asp Leu Val Val Asn His Thr Ser Asp Glu His 115 120 125 ThrTrp Phe Ile Glu Ala Arg Glu His Pro Asp Ser Ser Glu Arg Asp 130 135 140Tyr Tyr Ile Trp Cys Asp Gln Pro Asn Asp Leu Glu Ser Ile Phe Gly 145 150155 160 Gly Ser Ala Trp Gln Tyr Asp Asp Lys Ser Asp Gln Tyr Tyr Leu His165 170 175 Phe Phe Ser Lys Lys Gln Pro Asp Leu Asn Trp Glu Asn Ala AsnLeu 180 185 190 Arg Gln Lys Ile Tyr Asp Met Met Asn Phe Trp Ile Asp LysGly Ile 195 200 205 Gly Gly Phe Arg Met Asp Val Ile Asp Met Ile Gly LysIle Pro Ala 210 215 220 Gln His Ile Val Ser Asn Gly Pro Lys Leu His AlaTyr Leu Lys Glu 225 230 235 240 Met Asn Ala Ala Ser Phe Gly Gln His AspLeu Leu Thr Val Gly Glu 245 250 255 Thr Trp Gly Ala Thr Pro Glu Ile AlaLys Gln Tyr Ser Asn Pro Val 260 265 270 Asn His Glu Leu Ser Met Ile PheGln Phe Glu His Ile Gly Leu Gln 275 280 285 His Lys Pro Glu Ala Pro LysTrp Asp Tyr Val Lys Glu Leu Asn Val 290 295 300 Pro Ala Leu Lys Thr IlePhe Asn Lys Trp Gln Thr Glu Leu Glu Leu 305 310 315 320 Gly Gln Gly TrpAsn Ser Leu Phe Trp Asn Asn His Asp Leu Pro Arg 325 330 335 Val Leu SerIle Trp Gly Asn Thr Gly Lys Tyr Arg Glu Lys Ser Ala 340 345 350 Lys AlaLeu Ala Ile Leu Leu His Leu Met Arg Gly Thr Pro Tyr Ile 355 360 365 TyrGln Gly Glu Glu Ile Gly Met Thr Asn Tyr Pro Phe Lys Asp Leu 370 375 380Asn Glu Leu Asp Asp Ile Glu Ser Leu Asn Tyr Ala Lys Glu Ala Phe 385 390395 400 Thr Asn Gly Lys Ser Met Glu Thr Ile Met Asp Ser Ile Arg Met Ile405 410 415 Gly Arg Asp Asn Ala Arg Thr Pro Met Gln Trp Asp Ala Ser GlnAsn 420 425 430 Ala Gly Phe Ser Thr Ala Asp Lys Thr Trp Leu Pro Val AsnPro Asn 435 440 445 Tyr Lys Asp Ile Asn Val Gln Ala Ala Leu Lys Asn SerAsn Ser Ile 450 455 460 Phe Tyr Thr Tyr Gln Gln Leu Ile Gln Leu Arg LysGlu Asn Asp Trp 465 470 475 480 Leu Val Asp Ala Asp Phe Glu Leu Leu ProThr Ala Asp Lys Val Phe 485 490 495 Ala Tyr Leu Arg Lys Val Arg Glu GluArg Tyr Leu Ile Val Val Asn 500 505 510 Val Ser Asp Gln Glu Glu Val LeuGlu Ile Asp Val Asp Lys Gln Glu 515 520 525 Thr Leu Ile Ser Asn Thr AsnGlu Ser Ala Ala Leu Ala Asn His Lys 530 535 540 Leu Gln Pro Trp Asp AlaPhe Cys Ile Lys Ile Asn 545 550 555 36 1857 DNA Bacillus andBifidobacterium breve 36 atgagtttaa ccatcagtct gccgggtgtt caggctagcgcgatgaccgc caacaacctc 60 aatgacgact ggtggaagca ggccgtcgtt taccagatttacccgcgcag cttcaaggac 120 gttaacggcg acggcatcgg cgacatcgcc ggcgttaccgagaaaatgga ctacctgaaa 180 aacctcggcg tggacgccat ctggctctcc ccgttctacccctccgatct ggcggacggc 240 ggctacgacg tgatcgacta ccgcaacgtc gacccgcgactgggcaccat ggacgacttc 300 gacgccatgg ccaaagccgc gcatgaggcc ggcatcaaggtgatcgtgga catcgtgccc 360 aatcacaccg ccgacaagca cgtgttcttc aaggaagccctcgccgccga gcccggctcc 420 ccggcgcgcg accgctacat cttccgcgac ggccgcggcgagcacggcga actgccgccc 480 aacgactggc agtccttctt cggcggcccg gcctgggctcgcgtggccga cggccagtgg 540 tatctgcacc tgttcgacaa ggcgcaaccg gacgtcaactggaagaaccc ggacatccac 600 gaggaattca agaaaaccct gcgcttctgg tccgaccacggcaccgacgg cttccgcatc 660 gacgtggcgc acggtctggc caaagacctt gaatccaagccgctggagga gctcggccgc 720 gaatacagcg tggtcggcgt gctgaatcac gacttcagccatccgctgtt cgaccgccgc 780 gaagtgcacg acatctaccg cgaatggcgc aaggtgttcaacgagtacga cccgccgcgc 840 tttgccgtgg ccgaggcgtg ggtggtaccc gagcaccagcacctgtatgc ctcgatggat 900 gagctggggc agtccttcaa cttcgacttt gcgcaggccagctggtatgc cgatgagttc 960 cgcgcagcca tcgccgcggg tctcaaggcc gctgccgaaaccggcggttc caccaccacg 1020 tgggtgatga acaaccatga cgtgccgcgc agcccctcccgctatggtct accgcaggtc 1080 aagggcgcgc cttaccacca gctgccgcac gactggctgctgcgcaacgg caccacgtat 1140 cccgaggatc gcgagcttgg cacccgccgc gcccgcgccgccgctttgat ggagctcggc 1200 ctgcccggcg ccgcctatat ctatcagggc gaggagctgggcctgtttga agtggccgat 1260 attccgtggg atcgactgga agatccgacc gctttccacaccgctcaggc cacgatggac 1320 aagggccgag acggctgccg cgtgccgatt ccgtggaccgctgcaaacga accgaccttg 1380 gctgatttca gccgcccgat cccggccgat gacggcaccggcgagaacca cgtgccgctg 1440 tgcgccgccg gccagttcgg cacgggcgct tccttcggcttctcgccggc tacgcgcgct 1500 gagggcgtga cgccggccgc cgacccgcac ctgccgcagccgttgtggtt caaggattac 1560 gcggtggacg tggagcaggc cgacccggat tcaatgctcgcgctgtatca tgcggcgttg 1620 gcgattcgtc aggagtcgct gaccgccacg cgtgacaccaccgctgagca ggtggatatg 1680 gggccggacg tggtggccta cacccgcgcg gcggttggtggccgcacgtt cacctcgatc 1740 accaacttcg gcaccgagcc ggtggagctg cctggaggctccgtcgtgct gacgtccggc 1800 ccgctgaccc ccgacggcca gctccccacc gacacttctgcgtgggttat caagtag 1857 37 618 PRT Bacillus and Bifidobacterium breve 37Met Ser Leu Thr Ile Ser Leu Pro Gly Val Gln Ala Ser Ala Met Thr 1 5 1015 Ala Asn Asn Leu Asn Asp Asp Trp Trp Lys Gln Ala Val Val Tyr Gln 20 2530 Ile Tyr Pro Arg Ser Phe Lys Asp Val Asn Gly Asp Gly Ile Gly Asp 35 4045 Ile Ala Gly Val Thr Glu Lys Met Asp Tyr Leu Lys Asn Leu Gly Val 50 5560 Asp Ala Ile Trp Leu Ser Pro Phe Tyr Pro Ser Asp Leu Ala Asp Gly 65 7075 80 Gly Tyr Asp Val Ile Asp Tyr Arg Asn Val Asp Pro Arg Leu Gly Thr 8590 95 Met Asp Asp Phe Asp Ala Met Ala Lys Ala Ala His Glu Ala Gly Ile100 105 110 Lys Val Ile Val Asp Ile Val Pro Asn His Thr Ala Asp Lys HisVal 115 120 125 Phe Phe Lys Glu Ala Leu Ala Ala Glu Pro Gly Ser Pro AlaArg Asp 130 135 140 Arg Tyr Ile Phe Arg Asp Gly Arg Gly Glu His Gly GluLeu Pro Pro 145 150 155 160 Asn Asp Trp Gln Ser Phe Phe Gly Gly Pro AlaTrp Ala Arg Val Ala 165 170 175 Asp Gly Gln Trp Tyr Leu His Leu Phe AspLys Ala Gln Pro Asp Val 180 185 190 Asn Trp Lys Asn Pro Asp Ile His GluGlu Phe Lys Lys Thr Leu Arg 195 200 205 Phe Trp Ser Asp His Gly Thr AspGly Phe Arg Ile Asp Val Ala His 210 215 220 Gly Leu Ala Lys Asp Leu GluSer Lys Pro Leu Glu Glu Leu Gly Arg 225 230 235 240 Glu Tyr Ser Val ValGly Val Leu Asn His Asp Phe Ser His Pro Leu 245 250 255 Phe Asp Arg ArgGlu Val His Asp Ile Tyr Arg Glu Trp Arg Lys Val 260 265 270 Phe Asn GluTyr Asp Pro Pro Arg Phe Ala Val Ala Glu Ala Trp Val 275 280 285 Val ProGlu His Gln His Leu Tyr Ala Ser Met Asp Glu Leu Gly Gln 290 295 300 SerPhe Asn Phe Asp Phe Ala Gln Ala Ser Trp Tyr Ala Asp Glu Phe 305 310 315320 Arg Ala Ala Ile Ala Ala Gly Leu Lys Ala Ala Ala Glu Thr Gly Gly 325330 335 Ser Thr Thr Thr Trp Val Met Asn Asn His Asp Val Pro Arg Ser Pro340 345 350 Ser Arg Tyr Gly Leu Pro Gln Val Lys Gly Ala Pro Tyr His GlnLeu 355 360 365 Pro His Asp Trp Leu Leu Arg Asn Gly Thr Thr Tyr Pro GluAsp Arg 370 375 380 Glu Leu Gly Thr Arg Arg Ala Arg Ala Ala Ala Leu MetGlu Leu Gly 385 390 395 400 Leu Pro Gly Ala Ala Tyr Ile Tyr Gln Gly GluGlu Leu Gly Leu Phe 405 410 415 Glu Val Ala Asp Ile Pro Trp Asp Arg LeuGlu Asp Pro Thr Ala Phe 420 425 430 His Thr Ala Gln Ala Thr Met Asp LysGly Arg Asp Gly Cys Arg Val 435 440 445 Pro Ile Pro Trp Thr Ala Ala AsnGlu Pro Thr Leu Ala Asp Phe Ser 450 455 460 Arg Pro Ile Pro Ala Asp AspGly Thr Gly Glu Asn His Val Pro Leu 465 470 475 480 Cys Ala Ala Gly GlnPhe Gly Thr Gly Ala Ser Phe Gly Phe Ser Pro 485 490 495 Ala Thr Arg AlaGlu Gly Val Thr Pro Ala Ala Asp Pro His Leu Pro 500 505 510 Gln Pro LeuTrp Phe Lys Asp Tyr Ala Val Asp Val Glu Gln Ala Asp 515 520 525 Pro AspSer Met Leu Ala Leu Tyr His Ala Ala Leu Ala Ile Arg Gln 530 535 540 GluSer Leu Thr Ala Thr Arg Asp Thr Thr Ala Glu Gln Val Asp Met 545 550 555560 Gly Pro Asp Val Val Ala Tyr Thr Arg Ala Ala Val Gly Gly Arg Thr 565570 575 Phe Thr Ser Ile Thr Asn Phe Gly Thr Glu Pro Val Glu Leu Pro Gly580 585 590 Gly Ser Val Val Leu Thr Ser Gly Pro Leu Thr Pro Asp Gly GlnLeu 595 600 605 Pro Thr Asp Thr Ser Ala Trp Val Ile Lys 610 615 38 1653DNA Bacillus and Bifidobacterium breve 38 atgagtttaa ccatcagtctgccgggtgtt caggctagcg cgatgcaaaa acattggtgg 60 cacaaggcaa ctgtttatcaaatttatcca aaatctttta tggatacaaa tggtgatgga 120 attggtgatc tcaaaggtattacgagtaaa ttggattatt tgcaaaagtt aggggttatg 180 gctatttggc tatctccagtttatgatagc cccatggatg acaatggcta tgacattgcg 240 aactatgaag caattgcggatatttttggc aatatggctg atatggataa tttgctgacg 300 caggcaaaaa tgcgcgacataaaaatcatt atggatctag tggttaatca tacctcagat 360 gaacatactt ggtttattgaagcacgtgag catccagaca gttctgaacg cgattattat 420 atttggtgtg accagccaaatgatttggaa tctattttcg gtggttctgc ttggcagtat 480 gatgataagt ccgatcaatattatttgcat ttttttagta agaagcagcc agatctaaac 540 tgggaaaacg caaacttacgtcagaagatt tatgatatga tgaatttctg gattgataaa 600 ggtattggcg gctttcggatggacgtcatt gatatgattg ggaaaattcc tgctcagcat 660 attgtcagta acggaccaaaattgcatgct tatcttaagg agatgaatgc cgctagtttt 720 ggtcaacatg atctgctgactgtgggggaa acttggggag caacgcctga gattgcgaag 780 caatattcaa atccagtcaatcacgaactc tctatgattt ttcaatttga acatattggt 840 cttcagcata aaccagaagctcctaaatgg gattatgtga aggaacttaa tgttcctgct 900 ttaaaaacaa tctttaataaatggcagact gagttggaat taggacaggg gtggaattcg 960 ttattctgga ataaccatgacctgcctcgt gttttatcaa tctggggaaa tacgggcaaa 1020 tatcgtgaga agtctgctaaagcactggct attcttcttc accttatgcg tgggacacct 1080 tatatttatc aaggtgaagagattgggatg accaattatc cttttaaaga tttaaatgaa 1140 cttgatgata ttgaatcacttaattatgct aaggaagctt ttacaaatgg taagtctatg 1200 gaaactatca tggacagtattcgtatgatt ggccgtgata atgccagaac acctatgcaa 1260 tgggatgctt ctcaaaatgccggattttca acagcggata aaacatggct gccagttaat 1320 ccaaactata aagacatcaatgttcaagca gctctgaaaa attccaattc tatcttttac 1380 acctatcaac aactcattcagcttcgaaaa gaaaatgatt ggctagtaga tgccgatttt 1440 gaattgctcc ctacagcggacaaagtattt gcctatttac gaaaggtaag agaagaaagg 1500 tatcttatag tggtcaatgtttcagatcag gaagaagttc tagagattga tgttgacaaa 1560 caagaaactc tcattagcaatacaaatgaa agcgctgctc ttgccaatca caaactccag 1620 ccttgggatg ctttttgtattaagataaac taa 1653 39 550 PRT Bacillus and Bifidobacterium breve 39 MetSer Leu Thr Ile Ser Leu Pro Gly Val Gln Ala Ser Ala Met Gln 1 5 10 15Lys His Trp Trp His Lys Ala Thr Val Tyr Gln Ile Tyr Pro Lys Ser 20 25 30Phe Met Asp Thr Asn Gly Asp Gly Ile Gly Asp Leu Lys Gly Ile Thr 35 40 45Ser Lys Leu Asp Tyr Leu Gln Lys Leu Gly Val Met Ala Ile Trp Leu 50 55 60Ser Pro Val Tyr Asp Ser Pro Met Asp Asp Asn Gly Tyr Asp Ile Ala 65 70 7580 Asn Tyr Glu Ala Ile Ala Asp Ile Phe Gly Asn Met Ala Asp Met Asp 85 9095 Asn Leu Leu Thr Gln Ala Lys Met Arg Asp Ile Lys Ile Ile Met Asp 100105 110 Leu Val Val Asn His Thr Ser Asp Glu His Thr Trp Phe Ile Glu Ala115 120 125 Arg Glu His Pro Asp Ser Ser Glu Arg Asp Tyr Tyr Ile Trp CysAsp 130 135 140 Gln Pro Asn Asp Leu Glu Ser Ile Phe Gly Gly Ser Ala TrpGln Tyr 145 150 155 160 Asp Asp Lys Ser Asp Gln Tyr Tyr Leu His Phe PheSer Lys Lys Gln 165 170 175 Pro Asp Leu Asn Trp Glu Asn Ala Asn Leu ArgGln Lys Ile Tyr Asp 180 185 190 Met Met Asn Phe Trp Ile Asp Lys Gly IleGly Gly Phe Arg Met Asp 195 200 205 Val Ile Asp Met Ile Gly Lys Ile ProAla Gln His Ile Val Ser Asn 210 215 220 Gly Pro Lys Leu His Ala Tyr LeuLys Glu Met Asn Ala Ala Ser Phe 225 230 235 240 Gly Gln His Asp Leu LeuThr Val Gly Glu Thr Trp Gly Ala Thr Pro 245 250 255 Glu Ile Ala Lys GlnTyr Ser Asn Pro Val Asn His Glu Leu Ser Met 260 265 270 Ile Phe Gln PheGlu His Ile Gly Leu Gln His Lys Pro Glu Ala Pro 275 280 285 Lys Trp AspTyr Val Lys Glu Leu Asn Val Pro Ala Leu Lys Thr Ile 290 295 300 Phe AsnLys Trp Gln Thr Glu Leu Glu Leu Gly Gln Gly Trp Asn Ser 305 310 315 320Leu Phe Trp Asn Asn His Asp Leu Pro Arg Val Leu Ser Ile Trp Gly 325 330335 Asn Thr Gly Lys Tyr Arg Glu Lys Ser Ala Lys Ala Leu Ala Ile Leu 340345 350 Leu His Leu Met Arg Gly Thr Pro Tyr Ile Tyr Gln Gly Glu Glu Ile355 360 365 Gly Met Thr Asn Tyr Pro Phe Lys Asp Leu Asn Glu Leu Asp AspIle 370 375 380 Glu Ser Leu Asn Tyr Ala Lys Glu Ala Phe Thr Asn Gly LysSer Met 385 390 395 400 Glu Thr Ile Met Asp Ser Ile Arg Met Ile Gly ArgAsp Asn Ala Arg 405 410 415 Thr Pro Met Gln Trp Asp Ala Ser Gln Asn AlaGly Phe Ser Thr Ala 420 425 430 Asp Lys Thr Trp Leu Pro Val Asn Pro AsnTyr Lys Asp Ile Asn Val 435 440 445 Gln Ala Ala Leu Lys Asn Ser Asn SerIle Phe Tyr Thr Tyr Gln Gln 450 455 460 Leu Ile Gln Leu Arg Lys Glu AsnAsp Trp Leu Val Asp Ala Asp Phe 465 470 475 480 Glu Leu Leu Pro Thr AlaAsp Lys Val Phe Ala Tyr Leu Arg Lys Val 485 490 495 Arg Glu Glu Arg TyrLeu Ile Val Val Asn Val Ser Asp Gln Glu Glu 500 505 510 Val Leu Glu IleAsp Val Asp Lys Gln Glu Thr Leu Ile Ser Asn Thr 515 520 525 Asn Glu SerAla Ala Leu Ala Asn His Lys Leu Gln Pro Trp Asp Ala 530 535 540 Phe CysIle Lys Ile Asn 545 550 40 1860 DNA Bacillus and Bifidobacterium breve40 atgagtttaa ccatcagtct gccgggtgtt caggctagcg cgatgatgac ctctttcaac 60cgtgaacccc tgcccgacgc cgtccgcacg aatggcgcct ccccgaaccc gtggtggtcg 120aacgccgtcg tctaccagat ttacccacgt tccttccagg acacgaacgg cgatggtttc 180ggagatctta agggcattac ttcccgcctc gactatcttg ccgacctcgg cgtggatgtg 240ctgtggctct ccccggtcta caggtccccg caagacgaca acggctacga catctccgac 300taccgggaca tcgacccgct gttcggcacg ctcgacgaca tggacgagct gctcgccgaa 360gcgcacaagc gcggcctcaa gatcgtgatg gacctggtcg tcaaccacac ctccgacgag 420cacgcgtggt tcgaggcgtc gaaggacaag gacgacccgc acgccgactg gtactggtgg 480cgtcccgccc gccccggcca cgagcccggc acgcccggcg ccgagccgaa ccagtggggc 540tcctacttcg gcggctccgc atgggaatat tgccccgagc gtggtgagta ctatctccac 600cagttctcga agaagcagcc ggacctcaac tgggagaacc cggccgtacg ccgagccgtg 660tacgacatga tgaactggtg gctcgatcgc ggcatcgacg gcttccgcat ggacgtcatc 720accctgatct ccaagcgcac ggatgcaaac ggcaggctgc ccggcgagac cggttccgag 780ctgcaggacc tgccggtggg ggaggagggc tactccaacc cgaacccgtt ctgcgccgac 840ggtccgcgtc aggacgagtt cctcgccgag atgcgccgcg aggtgttcga cgggcgtgac 900ggcttcctca ccgtcggcga ggcccccggc atcaccgccg aacgcaacga gcacatcacc 960gaccccgcca acggcgagct ggatatgctc ttcctgttcg aacacatggg cgtcgaccaa 1020acccccgaat cgaaatggga cgacaaacca tggacgccgg ccgacctcga aaccaagctc 1080gccgaacaac aggacgccat cgcccgacac ggctgggcca gcctgttcct cgacaaccac 1140gaccagccgc gtgtcgtctc ccgttggggc gacgacacca gcaagaccgg ccgcatccgc 1200tccgccaagg cgctcgcgct gctgctgcac atgcaccgcg gcactccgta tgtctaccag 1260ggcgaggagc tcggcatgac caatgcgcac ttcacctcgc tcgaccagta ccgcgacctc 1320gaatccatca acgcctacca tcaacgcgtc gaggaaaccg ggatacggac atcggagacc 1380atgatgcgat ccctcgcccg atacggcagg gacaacgcgc gcaccccgat gcaatgggac 1440gactccacct acgccggctt caccatgccc gacgccccgg tcgaaccctg gatcgccgtc 1500aacccgaacc acacggagat caacgccgcc gacgagatcg acgaccccga ctccgtgtac 1560tcgttccaca aacggctcat cgccctgcgt cacaccgacc ccgtggtcgc cgccggcgac 1620taccgacgcg tggaaaccgg aaacgaccgg atcatcgcct tcaccagaac cctcgacgag 1680cgaaccatcc tcaccgtcat caacctctcg cccacacagg ccgcaccggc cggagaactg 1740gaaacgatgc ccgacggcac gatcctcatc gccaacacgg acgatcccgt aggaaacctg 1800aaaaccacgg gaacactcgg accatgggag gcgttcgcca tggaaaccga tccggaataa 186041 619 PRT Bacillus and Bifidobacterium breve 41 Met Ser Leu Thr Ile SerLeu Pro Gly Val Gln Ala Ser Ala Met Met 1 5 10 15 Thr Ser Phe Asn ArgGlu Pro Leu Pro Asp Ala Val Arg Thr Asn Gly 20 25 30 Ala Ser Pro Asn ProTrp Trp Ser Asn Ala Val Val Tyr Gln Ile Tyr 35 40 45 Pro Arg Ser Phe GlnAsp Thr Asn Gly Asp Gly Phe Gly Asp Leu Lys 50 55 60 Gly Ile Thr Ser ArgLeu Asp Tyr Leu Ala Asp Leu Gly Val Asp Val 65 70 75 80 Leu Trp Leu SerPro Val Tyr Arg Ser Pro Gln Asp Asp Asn Gly Tyr 85 90 95 Asp Ile Ser AspTyr Arg Asp Ile Asp Pro Leu Phe Gly Thr Leu Asp 100 105 110 Asp Met AspGlu Leu Leu Ala Glu Ala His Lys Arg Gly Leu Lys Ile 115 120 125 Val MetAsp Leu Val Val Asn His Thr Ser Asp Glu His Ala Trp Phe 130 135 140 GluAla Ser Lys Asp Lys Asp Asp Pro His Ala Asp Trp Tyr Trp Trp 145 150 155160 Arg Pro Ala Arg Pro Gly His Glu Pro Gly Thr Pro Gly Ala Glu Pro 165170 175 Asn Gln Trp Gly Ser Tyr Phe Gly Gly Ser Ala Trp Glu Tyr Cys Pro180 185 190 Glu Arg Gly Glu Tyr Tyr Leu His Gln Phe Ser Lys Lys Gln ProAsp 195 200 205 Leu Asn Trp Glu Asn Pro Ala Val Arg Arg Ala Val Tyr AspMet Met 210 215 220 Asn Trp Trp Leu Asp Arg Gly Ile Asp Gly Phe Arg MetAsp Val Ile 225 230 235 240 Thr Leu Ile Ser Lys Arg Thr Asp Ala Asn GlyArg Leu Pro Gly Glu 245 250 255 Thr Gly Ser Glu Leu Gln Asp Leu Pro ValGly Glu Glu Gly Tyr Ser 260 265 270 Asn Pro Asn Pro Phe Cys Ala Asp GlyPro Arg Gln Asp Glu Phe Leu 275 280 285 Ala Glu Met Arg Arg Glu Val PheAsp Gly Arg Asp Gly Phe Leu Thr 290 295 300 Val Gly Glu Ala Pro Gly IleThr Ala Glu Arg Asn Glu His Ile Thr 305 310 315 320 Asp Pro Ala Asn GlyGlu Leu Asp Met Leu Phe Leu Phe Glu His Met 325 330 335 Gly Val Asp GlnThr Pro Glu Ser Lys Trp Asp Asp Lys Pro Trp Thr 340 345 350 Pro Ala AspLeu Glu Thr Lys Leu Ala Glu Gln Gln Asp Ala Ile Ala 355 360 365 Arg HisGly Trp Ala Ser Leu Phe Leu Asp Asn His Asp Gln Pro Arg 370 375 380 ValVal Ser Arg Trp Gly Asp Asp Thr Ser Lys Thr Gly Arg Ile Arg 385 390 395400 Ser Ala Lys Ala Leu Ala Leu Leu Leu His Met His Arg Gly Thr Pro 405410 415 Tyr Val Tyr Gln Gly Glu Glu Leu Gly Met Thr Asn Ala His Phe Thr420 425 430 Ser Leu Asp Gln Tyr Arg Asp Leu Glu Ser Ile Asn Ala Tyr HisGln 435 440 445 Arg Val Glu Glu Thr Gly Ile Arg Thr Ser Glu Thr Met MetArg Ser 450 455 460 Leu Ala Arg Tyr Gly Arg Asp Asn Ala Arg Thr Pro MetGln Trp Asp 465 470 475 480 Asp Ser Thr Tyr Ala Gly Phe Thr Met Pro AspAla Pro Val Glu Pro 485 490 495 Trp Ile Ala Val Asn Pro Asn His Thr GluIle Asn Ala Ala Asp Glu 500 505 510 Ile Asp Asp Pro Asp Ser Val Tyr SerPhe His Lys Arg Leu Ile Ala 515 520 525 Leu Arg His Thr Asp Pro Val ValAla Ala Gly Asp Tyr Arg Arg Val 530 535 540 Glu Thr Gly Asn Asp Arg IleIle Ala Phe Thr Arg Thr Leu Asp Glu 545 550 555 560 Arg Thr Ile Leu ThrVal Ile Asn Leu Ser Pro Thr Gln Ala Ala Pro 565 570 575 Ala Gly Glu LeuGlu Thr Met Pro Asp Gly Thr Ile Leu Ile Ala Asn 580 585 590 Thr Asp AspPro Val Gly Asn Leu Lys Thr Thr Gly Thr Leu Gly Pro 595 600 605 Trp GluAla Phe Ala Met Glu Thr Asp Pro Glu 610 615 42 1653 DNA Bacillus andStreptococcus mutans 42 atgagtttaa ccatcagtct gccgggtgtt caggctagcgcgatgcaaaa acattggtgg 60 cacaaggcaa ctgtttatca aatttatcca aaatcttttatggatacaaa tggtgatgga 120 attggtgatc tcaaaggtat tacgagtaaa ttggattatttgcaaaagtt aggggttatg 180 gctatttggc tatctccagt ttatgatagc cccatggatgacaatggcta tgacattgcg 240 aactatgaag caattgcgga tatttttggc aatatggctgatatggataa tttgctgacg 300 caggcaaaaa tgcgcgacat aaaaatcatt atggatctagtggttaatca tacctcagat 360 gaacatactt ggtttattga agcacgtgag catccagacagttctgaacg cgattattat 420 atttggtgtg accagccaaa tgatttggaa tctattttcggtggttctgc ttggcagtat 480 gatgataagt ccgatcaata ttatttgcat ttttttagtaagaagcagcc agatctaaac 540 tgggaaaacg caaacttacg tcagaagatt tatgatatgatgaatttctg gattgataaa 600 ggtattggcg gctttcggat ggacgtcatt gatatgattgggaaaattcc tgctcagcat 660 attgtcagta acggaccaaa attgcatgct tatcttaaggagatgaatgc cgctagtttt 720 ggtcaacatg atctgctgac tgtgggggaa acttggggagcaacgcctga gattgcgaag 780 caatattcaa atccagtcaa tcacgaactc tctatgatttttcaatttga acatattggt 840 cttcagcata aaccagaagc tcctaaatgg gattatgtgaaggaacttaa tgttcctgct 900 ttaaaaacaa tctttaataa atggcagact gagttggaattaggacaggg gtggaattcg 960 ttattctgga ataaccatga cctgcctcgt gttttatcaatctggggaaa tacgggcaaa 1020 tatcgtgaga agtctgctaa agcactggct attcttcttcaccttatgcg tgggacacct 1080 tatatttatc aaggtgaaga gattgggatg accaattatccttttaaaga tttaaatgaa 1140 cttgatgata ttgaatcact taattatgct aaggaagcttttacaaatgg taagtctatg 1200 gaaactatca tggacagtat tcgtatgatt ggccgtgataatgccagaac acctatgcaa 1260 tgggatgctt ctcaaaatgc cggattttca acagcggataaaacatggct gccagttaat 1320 ccaaactata aagacatcaa tgttcaagca gctctgaaaaattccaattc tatcttttac 1380 acctatcaac aactcattca gcttcgaaaa gaaaatgattggctagtaga tgccgatttt 1440 gaattgctcc ctacagcgga caaagtattt gcctatttacgaaaggtaag agaagaaagg 1500 tatcttatag tggtcaatgt ttcagatcag gaagaagttctagagattga tgttgacaaa 1560 caagaaactc tcattagcaa tacaaatgaa agcgctgctcttgccaatca caaactccag 1620 ccttgggatg ctttttgtat taagataaac taa 1653 43550 PRT Bacillus and Streptococcus mutans 43 Met Ser Leu Thr Ile Ser LeuPro Gly Val Gln Ala Ser Ala Met Gln 1 5 10 15 Lys His Trp Trp His LysAla Thr Val Tyr Gln Ile Tyr Pro Lys Ser 20 25 30 Phe Met Asp Thr Asn GlyAsp Gly Ile Gly Asp Leu Lys Gly Ile Thr 35 40 45 Ser Lys Leu Asp Tyr LeuGln Lys Leu Gly Val Met Ala Ile Trp Leu 50 55 60 Ser Pro Val Tyr Asp SerPro Met Asp Asp Asn Gly Tyr Asp Ile Ala 65 70 75 80 Asn Tyr Glu Ala IleAla Asp Ile Phe Gly Asn Met Ala Asp Met Asp 85 90 95 Asn Leu Leu Thr GlnAla Lys Met Arg Asp Ile Lys Ile Ile Met Asp 100 105 110 Leu Val Val AsnHis Thr Ser Asp Glu His Thr Trp Phe Ile Glu Ala 115 120 125 Arg Glu HisPro Asp Ser Ser Glu Arg Asp Tyr Tyr Ile Trp Cys Asp 130 135 140 Gln ProAsn Asp Leu Glu Ser Ile Phe Gly Gly Ser Ala Trp Gln Tyr 145 150 155 160Asp Asp Lys Ser Asp Gln Tyr Tyr Leu His Phe Phe Ser Lys Lys Gln 165 170175 Pro Asp Leu Asn Trp Glu Asn Ala Asn Leu Arg Gln Lys Ile Tyr Asp 180185 190 Met Met Asn Phe Trp Ile Asp Lys Gly Ile Gly Gly Phe Arg Met Asp195 200 205 Val Ile Asp Met Ile Gly Lys Ile Pro Ala Gln His Ile Val SerAsn 210 215 220 Gly Pro Lys Leu His Ala Tyr Leu Lys Glu Met Asn Ala AlaSer Phe 225 230 235 240 Gly Gln His Asp Leu Leu Thr Val Gly Glu Thr TrpGly Ala Thr Pro 245 250 255 Glu Ile Ala Lys Gln Tyr Ser Asn Pro Val AsnHis Glu Leu Ser Met 260 265 270 Ile Phe Gln Phe Glu His Ile Gly Leu GlnHis Lys Pro Glu Ala Pro 275 280 285 Lys Trp Asp Tyr Val Lys Glu Leu AsnVal Pro Ala Leu Lys Thr Ile 290 295 300 Phe Asn Lys Trp Gln Thr Glu LeuGlu Leu Gly Gln Gly Trp Asn Ser 305 310 315 320 Leu Phe Trp Asn Asn HisAsp Leu Pro Arg Val Leu Ser Ile Trp Gly 325 330 335 Asn Thr Gly Lys TyrArg Glu Lys Ser Ala Lys Ala Leu Ala Ile Leu 340 345 350 Leu His Leu MetArg Gly Thr Pro Tyr Ile Tyr Gln Gly Glu Glu Ile 355 360 365 Gly Met ThrAsn Tyr Pro Phe Lys Asp Leu Asn Glu Leu Asp Asp Ile 370 375 380 Glu SerLeu Asn Tyr Ala Lys Glu Ala Phe Thr Asn Gly Lys Ser Met 385 390 395 400Glu Thr Ile Met Asp Ser Ile Arg Met Ile Gly Arg Asp Asn Ala Arg 405 410415 Thr Pro Met Gln Trp Asp Ala Ser Gln Asn Ala Gly Phe Ser Thr Ala 420425 430 Asp Lys Thr Trp Leu Pro Val Asn Pro Asn Tyr Lys Asp Ile Asn Val435 440 445 Gln Ala Ala Leu Lys Asn Ser Asn Ser Ile Phe Tyr Thr Tyr GlnGln 450 455 460 Leu Ile Gln Leu Arg Lys Glu Asn Asp Trp Leu Val Asp AlaAsp Phe 465 470 475 480 Glu Leu Leu Pro Thr Ala Asp Lys Val Phe Ala TyrLeu Arg Lys Val 485 490 495 Arg Glu Glu Arg Tyr Leu Ile Val Val Asn ValSer Asp Gln Glu Glu 500 505 510 Val Leu Glu Ile Asp Val Asp Lys Gln GluThr Leu Ile Ser Asn Thr 515 520 525 Asn Glu Ser Ala Ala Leu Ala Asn HisLys Leu Gln Pro Trp Asp Ala 530 535 540 Phe Cys Ile Lys Ile Asn 545 550

What is claimed is:
 1. An isolated nucleic acid molecule encoding anα(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide selectedfrom the group consisting of: (a) an isolated nucleic acid moleculeencoding the amino acid sequence SEQ ID NOs:2, 4, or 6; (b) a nucleicacid molecule that hybridizes with (a) under the following hybridizationconditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDSfollowed by 0.1×SSC, 0.1% SDS; and (c) a nucleic acid molecule that iscomplementary to (a) or (b).
 2. The isolated nucleic acid molecule ofclaim 1 selected from the group of nucleic acid molecules consisting ofSEQ ID NOs:1, 3, and
 5. 3. A polypeptide encoded by the isolated nucleicacid molecule of claim
 1. 4. The polypeptide of claim 3 selected fromthe group consisting of SEQ ID NOs:2, 4, and
 6. 5. An isolated nucleicacid molecule encoding an α(1,6)-linked glucose oligosaccharidehydrolyzing polypeptide selected from the group consisting of: (a) anisolated nucleic acid molecule encoding a chimeric protein comprised ofa signal peptide operably linked to an α(1,6)-linked glucoseoligosaccharide hydrolyzing polypeptide; (b) a nucleic acid moleculethat hybridizes with (a) under the following hybridization conditions:0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS; and (c) a nucleic acid molecule that is complementaryto (a) or (b).
 6. The isolated nucleic acid molecule of claim 5, whereinthe signal peptide is SEQ ID NO:24 or SEQ ID NO:25.
 7. The isolatednucleic acid molecule of claim 5, wherein the α(1,6)-linked glucoseoligosaccharide hydrolyzing polypeptide is SEQ ID NOs:2, 4, 6, 17, or31.
 8. The isolated nucleic acid molecule of claim 5, wherein the signalpeptide is SEQ ID NO:24 or SEQ ID NO:25, and wherein the α(1,6)-linkedglucose oligosaccharide hydrolyzing polypeptide is SEQ ID NOs:2, 4, 6,17, or
 31. 9. The isolated nucleic acid molecule of claim 5, wherein thesignal peptide is encoded by the signal sequence as set forth in SEQ IDNO:26 or SEQ ID NO:27.
 10. The isolated nucleic acid molecule of claim 5encoding the α(1,6)-linked glucose oligosaccharide hydrolyzingpolypeptide, the isolated nucleic acid molecule having the sequence asset forth in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, or SEQ ID NO:30.11. The isolated nucleic acid molecule of claim 5 selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:28, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42. 12.The polypeptide encoded by the nucleic acid molecule of claim
 5. 13. Thepolypeptide encoded by the isolated nucleic acid molecule of claim 9,claim 10, or claim
 11. 14. The polypeptide of claim 13 selected from thegroup consisting of SEQ ID NO:4, SEQ ID NO:29, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, and SEQ ID NO:43.
 15. Achimeric gene comprising the isolated nucleic acid molecule of claim 1or claim 5 operably linked to suitable regulatory sequences.
 16. Thechimeric gene of claim 15 wherein the suitable regulatory sequence isselected from the group comprising CYC1, HIS3, GAL1, GAL10, ADH1, PGK,PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, AOX1, lac, ara, tet, trp,IP_(L), IP_(R), T7, tac, trc, apr, npr, nos, and GI.
 17. A vectorcomprising the chimeric gene of claim
 15. 18. A transformed host cellcomprising the chimeric gene of claim
 15. 19. The transformed host cellof claim 18 wherein the chimeric gene is integrated into the chromosomeor is plasmid-borne.
 20. The transformed host cell of claim 18, whereinthe host cell is selected from the group consisting of bacteria, yeast,and filamentous fungi.
 21. The transformed host cell of claim 20,wherein the transformed host cell is selected from the generaAspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula,Rhodococcus, Acinetobacter, Arthrobacter, Brevibacterium, Acidovorax,Bacillus, Streptomyces, Escherichia, Salmonella, Pseudomonas, orCornyebacterium.
 22. The transformed host cell of claim 20 wherein thetransformed host cell is E. coli.
 23. A method for the production of atarget molecule in a recombinant host cell comprising: (a) contacting atransformed host cell comprising: (i) an isolated nucleic acid moleculeencoding a chimeric protein comprised of a signal peptide operablylinked to an α(1,6)-linked glucose oligosaccharide hydrolyzingpolypeptide; (ii) a nucleic acid molecule that hybridizes with (i) underthe following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. andwashed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or (iii) anucleic acid molecule that is complementary to (i) or (ii); and (iv) atleast one chimeric gene for converting mononsaccharides to the targetmolecule, in the presence of limit dextrin under suitable conditionswhereby the target molecule is produced; and (b) optionally recoveringthe target molecule produced in (a).
 24. A method for the production ofglycerol in a recombinant host cell comprising: (a) contacting atransformed host cell comprising: (i) an isolated nucleic acid moleculeencoding a chimeric protein comprised of a signal peptide operablylinked to an α(1,6)-linked glucose oligosaccharide hydrolyzingpolypeptide; (ii) a nucleic acid molecule that hybridizes with (i) underthe following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. andwashed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or (iii) anucleic acid molecule that is complementary to (i) or (ii); and (iv) atleast one chimeric gene for converting mononsaccharides to glycerol, inthe presence of limit dextrin under suitable conditions whereby glycerolis produced; and (b) optionally recovering the glycerol produced in (a).25. A method for the production of 1,3-propanediol in a recombinant hostcell comprising: (a) contacting a transformed host cell comprising: (i)an isolated nucleic acid molecule encoding a chimeric protein comprisedof a signal peptide operably linked to an α(1,6)-linked glucoseoligosaccharide hydrolyzing polypeptide; (ii) a nucleic acid moleculethat hybridizes with (i) under the following hybridization conditions:0.1×SSC, 0.1% SES, 65° C. and washed with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS; or (iii) a nucleic acid molecule that iscomplementary to (i) or (ii), (iv) at least one chimeric gene forconverting mononsaccharides to 1,3-propanediol, in the presence of limitdextrin under suitable conditions whereby 1,3-propanediol is produced;and (b) optionally recovering the 1,3-propanediol produced in (a).
 26. Amethod for the production of cell mass in a recombinant host cellcomprising: (a) contacting a transformed host cell comprising: (i) anisolated nucleic acid molecule encoding a chimeric protein comprised ofa signal peptide linked to an α(1,6)-linked glucose oligosaccharidehydrolyzing polypeptide; (ii) a nucleic acid molecule that hybridizeswith (i) under the following hybridization conditions: 0.1×SSC, 0.1%SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1%SDS; and (iii) a nucleic acid molecule that is complementary to (i) or(ii). under suitable conditions in the presence of limit dextrin; (b)optionally recovering the cell mass produced in (a).
 27. The method ofclaim 23, claim 24, claim 25 or claim 26 wherein the signal peptidecomprises SEQ ID NO:24 or SEQ ID NO:25.
 28. A method for the productionof a target molecule in a recombinant host cell comprising: (a)contacting a transformed host cell comprising: (i) an isolated nucleicacid molecule encoding the amino acid sequence selected from the groupconsisting of SEQ ID NOs:2, 6, 17 and 31; (ii) a nucleic acid moleculethat hybridizes with (i) under the following hybridization conditions:0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS; or (iii) a nucleic acid molecule that iscomplementary to (i) or (ii); and (iv) at least one chimeric gene forconverting mononsaccharides to the target molecule, in the presence oflimit dextrin under suitable conditions whereby the target molecule isproduced; and (b) optionally recovering the target molecule produced in(a).
 29. A method for the production of 1,3-propanediol in a recombinanthost cell comprising: (a) contacting a transformed host cell comprising:(i) an isolated nucleic acid molecule encoding the amino acid sequenceselected from the group consisting of SEQ ID NOs:2, 6, 17 and 31; (ii) anucleic acid molecule that hybridizes with (i) under the followinghybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or (iii) a nucleic acidmolecule that is complementary to (i) or (ii); and (iv) at least onechimeric gene for converting mononsaccharides to 1,3-propanediol; in thepresence of limit dextrin under suitable conditions whereby1,3-propanediol is produced; and (b) optionally recovering the1,3-propanediol produced in (a).
 30. A method for the production ofglycerol in a recombinant host cell comprising: (a) contacting atransformed host cell comprising: (i) an isolated nucleic acid moleculeencoding the amino acid sequence selected from the group consisting ofSEQ NOs:2, 6, 17 and 31; (ii) a nucleic acid molecule that hybridizeswith (i) under the following hybridization conditions: 0.1×SSC, 0.1%SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1%SDS; or (iii) a nucleic acid molecule that is complementary to (i) or(ii); and (iv) at least one chimeric gene for convertingmononsaccharides to glycerol; in the presence of limit dextrin undersuitable conditions whereby glycerol is produced; and (b) optionallyrecovering the glycerol produced in (a).
 31. A method for the productionof cell mass in a recombinant host cell comprising: (a) contacting atransformed host cell comprising: (i) an isolated nucleic acid moleculeencoding the amino acid sequence selected from the group consisting ofSEQ ID NOs:2, 6, 17 and 31; (ii) a nucleic acid molecule that hybridizeswith (i) under the following hybridization conditions: 0.1×SSC, 0.1%SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1%SDS; or (iii) a nucleic acid molecule that is complementary to (i) or(ii), in the presence of limit dextrin under suitable conditions wherebycell mass is produced; and (b) optionally recovering the cell massproduced in (a).
 32. The method of claim 28, claim 29, claim 30 or claim31 wherein the signal peptide is SEQ ID NO:24 or SEQ ID NO:25.
 33. Amethod for degrading limit dextrin comprising: (a) contacting atransformed host cell comprising: (i) a nucleic acid molecule encodingthe enzymes selected from the group consisting of SEQ ID NOs:2, 6, 17and 31; (ii) a nucleic acid molecule that hybridizes with (i) under thefollowing hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washedwith 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; or (iii) a nucleicacid molecule that is complementary to (i) or (ii), with an effectiveamount of limit dextrin substrate under suitable growth conditions; and(b) optionally recovering the product of step (a).
 34. A polypeptidehaving an amino acid sequence that has at least 69% identity based onthe BLASTP method of alignment when compared to a polypeptide having thesequence as set forth in SEQ ID NO:17, the polypeptide havingα(1,6)-linked glucose oligosaccharide hydrolyzing activity.